Post-translational modification directs nuclear and hyphal tip localization of Candida albicans mRNA-binding protein Slr1.

The morphological transition of the opportunistic fungal pathogen Candida albicans from budding to hyphal growth has been implicated in its ability to cause disease in animal models. Absence of SR-like RNA-binding protein Slr1 slows hyphal formation and decreases virulence in a systemic candidiasis model, suggesting a role for post-transcriptional regulation in these processes. SR (serine-arginine)-rich proteins influence multiple steps in mRNA metabolism and their localization and function are frequently controlled by modification. We now demonstrate that Slr1 binds to polyadenylated RNA and that its intracellular localization is modulated by phosphorylation and methylation. Wildtype Slr1-GFP is predominantly nuclear, but also co-fractionates with translating ribosomes. The non-phosphorylatable slr1-6SA-GFP protein, in which six serines in SR/RS clusters are substituted with alanines, primarily localizes to the cytoplasm in budding cells. Intriguingly, hyphal cells display a slr1-6SA-GFP focus at the tip near the Spitzenkörper, a vesicular structure involved in molecular trafficking to the tip. The presence of slr1-6SA-GFP hyphal tip foci is reduced in the absence of the mRNA-transport protein She3, suggesting that unphosphorylated Slr1 associates with mRNA-protein complexes transported to the tip. The impact of SLR1 deletion on hyphal formation and function thus may be partially due to a role in hyphal mRNA transport.

Introduction

Candida albicans is a common n class="Species">human commensal fungus as well as an opportunistic pathogen that can cause a wide range of diseases from relatively mild mucosal infections to systemic infections with mortality rates up to 37% (Wisplinghoff et al., 2004). C. albicans pathogenicity is linked to a switch between budding yeast and filamentous hyphal morphologies (Lo et al., 1997; Saville et al., 2003). While the compact yeast form may facilitate dissemination in the bloodstream, interaction of yeast cells with host epithelia leads to a transition to the hyphal form, which increases host–cell adherence and promotes invasion into host tissues (Filler et al., 1995; Dalle et al., 2010). The yeast‐to‐hyphal transition is accompanied by many changes in gene expression that facilitate invasive infection such as the upregulation of cell‐surface adhesins and secreted hydrolases (De Groot et al., 2013; Schaller et al., 2005). Whereas the signaling molecules and transcription factors required for this transition have been studied intensively (Nantel et al., 2002; Kadosh and Johnson, 2005; Whiteway and Bachewich, 2007; Bruno et al., 2010; Sellam et al., 2010), much less is known about proteins with roles in post‐transcriptional events that could influence hyphal development and function.

Post‐transcriptional processes are critical for cellular differentiation in diverse eukaryotic systems, from mRNA transport and turnover during n class="Species">Drosophila embryogenesis (Lasko, 2011), to splicing during meiosis in Saccharomyces cerevisiae (Spingola and Ares, 2000), to hyphal mRNA transport during filamentation of the corn smut fungus Ustilago maydis (Becht et al., 2005, 2006). The eukaryotic serine–arginine (SR) family of RNA‐binding proteins has numerous roles in the control of gene expression, from ubiquitous roles in splicing to impacts on mRNA transport, translation and stability (Shepard and Hertel, 2009; Long and Caceres, 2009; Zhong et al., 2009) and SR protein activity can influence metazoan cellular differentiation (Baker et al., 1989; Sen et al., 2013). SR proteins are characterized by the presence of one or two RNA‐recognition motifs (RRMs) and clusters of serine–arginine/arginine–serine (SR/RS) dipeptides. SR protein function and localization are frequently modulated by phosphorylation of the SR/RS dipeptides (Long and Caceres, 2009). While Schizosaccharomyces pombe has SR proteins (Srp1 and Srp2), neither S. cerevisiae nor C. albicans has canonical SR proteins; rather, these yeasts encode SR‐like proteins that also include one or two RRMs, but which have repetitive arginine (R)‐rich domains with fewer SR/RS dipeptides (Plass et al., 2008). These R‐rich domains are often targets for arginine methylation (Henry and Silver, 1996; McBride et al., 2007; Ariyachet et al., 2013). The most thoroughly studied yeast SR‐like protein, S. cerevisiae Npl3, has been implicated in many aspects of mRNA metabolism from transcription and splicing to mRNA export and translation (Kadowaki et al., 1994; Lee et al., 1996; Bucheli and Buratowski, 2005; Dermody et al., 2008; Kress et al., 2008; Baierlein et al., 2013). Both ScNpl31 and its S. pombe ortholog, SpSrp2, are essential, whereas SpSrp1 is not essential (Bossie et al., 1992; Gross et al., 1998; Lutzelberger et al., 1999).

Our recent work identified a C. albicans n class="Chemical">SR‐like RNA‐binding protein, Slr1, that lacks an apparent ortholog in S. cerevisiae (Ariyachet et al., 2013). Absence of this C. albicans SpSrp1 ortholog decreases C. albicans growth rate more than deletion of the ScNPL3/SpSRP2 ortholog CaNPL3. SLR1 deletion slows hyphal formation, leads to a defect in host cell damage in vitro and lowers virulence in a murine model of disseminated candidiasis (Ariyachet et al., 2013). Absence of Slr1 also increases exposure of hyphal‐specific adhesin Als3 on the cell surface, suggesting the importance of Slr1 for proper hyphal structure (Ariyachet et al., 2013).

Given the importance of Slr1 for C. albicans hyphal formation and virulence and its sequence similarity to other n class="Species">yeast SR and SR‐like proteins, we sought to address whether the sequence similarities reflect functional similarities, including identifying the molecular interactions and modifications of Slr1 and testing the impact of post‐translational modifications on Slr1 intracellular localization. Interestingly, we find that C‐terminal S‐to‐A mutations that block Slr1 phosphorylation also cause Slr1 to accumulate not only in the cytoplasm of yeast‐form cells, but also at the hyphal tip in a region close to the Spitzenkörper, a vesicular structure involved in trafficking lipids and proteins to the tip. The hyphal tip localization of mutant Slr1 is also partially dependent on the presence of mRNA transport protein She3. Combined with additional evidence that wildtype Slr1 interacts with polyadenylated RNA and mRNA‐binding proteins, these results suggest a model in which unphosphorylated Slr1 associates with mRNA–protein complexes transported to the hyphal tip where the mRNA is locally translated and that phosphorylation facilitates release of Slr1 from such transport complexes. In fact, Slr1 is detected in polysomes, supporting a potential role in translation. The impact of SLR1 deletion on hyphal formation and function thus may be due in part to a role in hyphal mRNA transport and translation.

Results

Structural and functional similarity of Slr1 and SR‐like proteins

The amino acid sequence of C. albicans Slr1 shows distinct similarity to that of other fungal n class="Chemical">SR‐like proteins, including the presence of an RNA‐recognition motif (RRM) N‐terminal to an arginine (R)‐rich region (Fig. 1A). BLASTP searches with the RRM of Slr1 revealed its similarity to RRMs of SR‐like proteins S. pombe Srp1 and Aspergillus nidulans SwoK (Fig. 1B); similar RRMs were also identified in other fungi from Lachancea thermotolerans to Ustilago maydis (Supporting Information Fig. S1), but not in S. cerevisiae. A motif common to many metazoan SR proteins that overlaps with RNP‐1 (EFEDxRDAEDA), however, is better conserved in AnSwoK and SpSrp1 than in CaSlr1 (Fig. 1B, boldface). The amino acid composition within the low complexity R‐rich region also differs among these proteins: RG dipeptides predominate in Slr1, RD dipeptides in AnSwoK and RS dipeptides in SpSrp1 (Fig. 1C). In addition, AnSwoK and SpSrp1 have regions C‐terminal to this R‐rich domain (Fig. 1A and C). Thus, the RRM is more highly conserved among Slr1‐related proteins than the C‐terminal region.

Figure 1

C. albicans Slr1 sequence similarity to fungal SR‐like proteins.

A. Domain comparison of S. pombe Srp1, A. nidulans SwoK, C. albicans Slr1 and S. cerevisiae Npl3. RNA‐recognition motifs (RRM) and arginine‐rich (R‐rich) domains, as well as non‐conserved N‐ and C‐terminal domains, are shown.

B. ClustalW sequence alignment of Slr1, AnSwoK and SpSrp1 RRM domains. Conserved RNP‐2 and RNP‐1 motifs are indicated. Residues in boldface within the boxed region are identical to a conserved motif found in metazoan SR proteins (Birney et al., 1993). The 5′ splice site for the first intron in SpSRP1, AnswoK and SLR1 genes is located after the second nucleotide in the codon for the underlined residue. Identical (*) and conserved (:) residues are indicated.

C. Comparison of arginine‐rich C‐termini. C‐terminal sequences starting at the first arginine following the RRM are shown. Arginine‐rich (boldface) regions, SR/RS dipeptides (solid underlining) and a C‐terminal SR/RS‐containing heptapeptide (dashed underlining) are indicated. Note the similarity between ScNpl3 and Slr1 C‐termini. Amino acids deleted in mutant Slr1 proteins are marked with brackets [slr1Δ151‐192] and parentheses (slr1Δ123‐194); asterisks mark serines mutated to alanine in slr1‐6SA.

D. Partial functionality of the C. albicans Slr1 C‐terminus in S. cerevisiae Npl3. S. cerevisiae cells lacking chromosomal NPL3 and bearing a ScNPL3 URA3 CEN plasmid were transformed with LEU2 plasmids expressing chimeric forms of ScNpl3 or the vector plasmid without NPL3. White boxes indicate ScNpl3 domains; black boxes indicate equivalent CaSlr1 domains that are replaced within ScNpl3 (C=C‐terminus). To test for chimeric ScNpl3 function, cells were grown to mid‐log‐phase and serial ten‐fold dilutions plated on medium lacking leucine and containing 5‐FOA (selecting for loss of the ScNPL3 URA3 plasmid). Cells were also plated on medium lacking leucine and uracil to confirm equal cell numbers. Plates were incubated at 30°C for 2 days prior to imaging.

C. albicans Slr1 sequence similarity to fungal n class="Chemical">SR‐like proteins.

A. Domain comparison of S. pombe n class="Gene">Srp1, A. nidulans SwoK, C. albicans Slr1 and S. cerevisiae Npl3. RNA‐recognition motifs (RRM) and arginine‐rich (R‐rich) domains, as well as non‐conserved N‐ and C‐terminal domains, are shown.

B. ClustalW sequence alignment of Slr1, AnSwoK and SpSrp1 RRM domains. Conserved RNP‐2 and RNP‐1 motifs are indicated. Residues in boldface within the boxed region are identical to a conserved motif found in metazoan n class="Chemical">SR proteins (Birney et al., 1993). The 5′ splice site for the first intron in SpSRP1, AnswoK and SLR1 genes is located after the second nucleotide in the codon for the underlined residue. Identical (*) and conserved (:) residues are indicated.

C. Comparison of arginine‐rich C‐termini. C‐terminal sequences starting at the first n class="Chemical">arginine following the RRM are shown. Arginine‐rich (boldface) regions, SR/RS dipeptides (solid underlining) and a C‐terminal SR/RS‐containing heptapeptide (dashed underlining) are indicated. Note the similarity between ScNpl3 and Slr1 C‐termini. Amino acids deleted in mutant Slr1 proteins are marked with brackets [slr1Δ151‐192] and parentheses (slr1Δ123‐194); asterisks mark serines mutated to alanine in slr1‐6SA.

D. Partial functionality of the C. albicans Slr1 C‐terminus in n class="Species">S. cerevisiae Npl3. S. cerevisiae cells lacking chromosomal NPL3 and bearing a ScNPL3 URA3 CEN plasmid were transformed with LEU2 plasmids expressing chimeric forms of ScNpl3 or the vector plasmid without NPL3. White boxes indicate ScNpl3 domains; black boxes indicate equivalent CaSlr1 domains that are replaced within ScNpl3 (C=C‐terminus). To test for chimeric ScNpl3 function, cells were grown to mid‐log‐phase and serial ten‐fold dilutions plated on medium lacking leucine and containing 5‐FOA (selecting for loss of the ScNPL3 URA3 plasmid). Cells were also plated on medium lacking leucine and uracil to confirm equal cell numbers. Plates were incubated at 30°C for 2 days prior to imaging.

Whereas S. cerevisiae does not encode a protein with an Slr1‐like RRM, the predominance of n class="Chemical">glycine and bulky hydrophobic residues in the R‐rich region of Slr1 does resemble this region of S. cerevisiae Npl3 (Fig. 1C). In addition, the C‐terminus of ScNpl3 (ReRSPtR) (Fig.1C), which influences cellular localization of ScNpl3 through its phosphorylation (Yun and Fu, 2000; Gilbert et al., 2001), resembles that of Slr1 (RsRSPeRy). This region is critical for ScNpl3 function: deletion of the R‐rich region and C‐terminus severely abrogates S. cerevisiae growth (McBride et al., 2009). Therefore, to determine whether this sequence similarity is functionally relevant, the ability of the R‐rich region of Slr1 to substitute for that of ScNpl3 was tested. As shown in Fig. 1D, chimeric ScNpl3 proteins bearing the Slr1 R‐rich domain supported growth of S. cerevisiae cells lacking Npl3 (npl3Δ). The Slr1 R‐rich region linked to the ScNpl3 C‐terminus supported only slightly more growth of npl3Δ cells than when it was linked to the Slr1 C‐terminus (Fig. 1D, compare rows 3 and 4). These results suggest that the Slr1 R‐rich region can mediate similar molecular interactions to those mediated by the ScNpl3 R‐rich region, but that these regions of the two proteins are not functionally identical.

Slr1 is present in mRNPs

Given the structural similarity of Slr1 to proteins with known roles in mRNA metabolism, we tested whether Slr1 interacts physically with polyadenylated mRNA and mRNA‐binding proteins. To assess whether Slr1 associates with poly(A) RNA, cells expressing Slr1 with a C‐terminal tandem affinity pn class="Chemical">urification (TAP) tag were exposed to UV light to crosslink RNA with bound proteins. Poly(A) RNAs were purified from cell lysates through two rounds of binding to oligo(dT) sepharose. Proteins bound to poly(A) RNAs were released by RNase treatment and Slr1‐TAP was detected by anti‐Protein A immunoblot. As shown in Fig. 2A, Slr1‐TAP did co‐purify when crosslinked to poly(A) RNA. In contrast, Slr1‐TAP was not purified from untreated cell lysates (Fig. 2A), indicating that Slr1‐TAP does not bind non‐specifically to the oligo (dT) resin. Therefore, C. albicans Slr1 associates with poly(A) RNA and is likely an mRNA‐binding protein.

Figure 2

Slr1 binds to mRNA.

A. Slr1‐TAP binds to polyadenylated RNA. Cells expressing Slr1‐TAP (SLR1‐TAP/slr1Δ) were exposed to UV light to crosslink RNA with bound proteins. Following lysis, polyadenylated RNA was isolated through two rounds of isolation with oligo(dT)‐sepharose. Proteins were released from the RNA by RNase digestion and resolved by SDS‐PAGE; the presence of Slr1‐TAP was determined by anti‐Protein A (PrA) immunoblotting. Samples not exposed to UV light were processed in parallel to test for non‐specific binding of Slr1‐TAP to the oligo(dT) resin.

B. Cap‐binding complex protein 1 (Cbc1; Orf19.387) co‐precipitates with Slr1‐TAP. Protein lysates (65 mg total protein) from C. albicans strains expressing untagged Slr1 (SLR1/slr1Δ) or Slr1‐TAP (SLR1‐TAP/slr1Δ) were incubated with IgG beads. Bound proteins were eluted with TEV protease and resolved by SDS‐10% PAGE. Proteins were visualized by zinc staining and identified by mass spectrometry.

C. RNA dependence of Slr1‐cap‐binding complex interaction. Protein lysates (5 mg total protein) were prepared from cells expressing Slr1‐TAP and Cbc1‐GFP (lanes 2–3; ORF19.387‐GFP/ORF19.387 SLR1‐TAP/slr1Δ) or Cbc2‐GFP (lanes 5–6; ORF19.763‐GFP/ORF19.763 SLR1‐TAP/slr1Δ). Lysates were either treated with RNase (lane 3, 6) or not treated (lane 2, 5) before IgG bead incubation. Slr1‐TAP‐bound proteins were eluted and analyzed by anti‐GFP immunoblotting. Lysates from Cbc‐GFP‐expressing strains that did not express Slr1‐TAP (ORF19.387‐GFP/ORF19.387 SLR1/slr1Δ and ORF19.763‐GFP/ORF19.763 SLR1/slr1Δ) were also incubated with IgG beads to test for non‐specific binding of Cbc‐GFP proteins to the beads (Cbc1, lane 1; Cbc2, lane 4).

A. Slr1‐TAP binds to polyadenylated RNA. Cells expressing Slr1‐TAP (SLR1‐TAP/slr1Δ) were exposed to UV light to crosslink RNA with bound proteins. Following lysis, polyadenylated RNA was isolated through two rounds of isolation with oligo(dT)‐n class="Chemical">sepharose. Proteins were released from the RNA by RNase digestion and resolved by SDS‐PAGE; the presence of Slr1‐TAP was determined by anti‐Protein A (PrA) immunoblotting. Samples not exposed to UV light were processed in parallel to test for non‐specific binding of Slr1‐TAP to the oligo(dT) resin.

B. Cap‐binding complex protein 1 (Cbc1; Orf19.387) co‐precipitates with Slr1‐TAP. Protein lysates (65 mg total protein) from C. albicans strains expressing untagged Slr1 (SLR1/slr1Δ) or Slr1‐TAP (SLR1‐TAP/slr1Δ) were incubated with IgG beads. Bound proteins were eluted with TEV protease and resolved by n class="Chemical">SDS‐10% PAGE. Proteins were visualized by zinc staining and identified by mass spectrometry.

C. RNA dependence of Slr1‐cap‐binding complex interaction. Protein lysates (5 mg total protein) were prepared from cells expressing Slr1‐TAP and Cbc1‐GFP (lanes 2–3; ORF19.387‐GFP/ORF19.387 SLR1‐TAP/slr1Δ) or Cbc2‐GFP (lanes 5–6; ORF19.763‐GFP/ORF19.763 SLR1‐TAP/slr1Δ). Lysates were either treated with RNase (lane 3, 6) or not treated (lane 2, 5) before IgG bead incubation. Slr1‐TAP‐bound proteins were eluted and analyzed by anti‐GFP immunoblotting. Lysates from Cbc‐GFP‐expressing strains that did not express Slr1‐TAP (ORF19.387‐GFP/ORF19.387 SLR1/slr1Δ and ORF19.763‐GFP/ORF19.763 SLR1/slr1Δ) were also incubated with IgG beads to test for non‐specific binding of Cbc‐GFP proteins to the beads (Cbc1, lane 1; n class="Gene">Cbc2, lane 4).

To test whether Slr1 interacts with known RNA‐binding proteins, we isolated proteins bound to Slr1‐TAP from lysates of yeast‐form n class="Species">C. albicans cells by purification on IgG‐sepharose and subsequent elution by cleavage of the tag with tobacco etch virus (TEV) protease (Fig. 2B). Lysates from cells expressing untagged Slr1 were used to detect background binding to the beads. Zinc staining of purified proteins revealed two major proteins that co‐purify specifically with Slr1‐TAP (Fig. 2B). Mass spectrometric analysis identified the lower band as Slr1‐calmodulin‐binding protein, the expected product following cleavage of the TAP‐tagged protein from the IgG beads. The other specific protein (∼100 kDa) corresponded to the C. albicans ortholog of Cbc1/Cbp80, the large subunit of the nuclear mRNA cap‐binding complex (CBC) (Lewis et al., 1996).

To confirm the interaction of Slr1 with the nuclear cap‐binding complex, large and small subunits of the complex were tagged with green fluorescent protein (GFP) in the Slr1‐TAP‐expressing strain. Anti‐GFP immunoblots following IgG purification of Slr1‐TAP from these strains supported an interaction between Slr1 and the CBC (Fig. 2C). The mRNA‐binding activity of Slr1 (Fig. 2A) suggested that the co‐pn class="Chemical">urification of the cap‐binding complex proteins might be due to simultaneous binding of Slr1 and the CBC to the same mRNA. Consistent with this model, RNase treatment of lysates prior to Slr1‐TAP isolation eliminated co‐purification of the Cbc‐GFP proteins (Fig. 2C). Slr1 therefore interacts indirectly with Cbc proteins in an RNA‐dependent manner, supporting the conclusion that Slr1 can interact with mRNA.

The C‐terminus of Slr1 influences its subcellular localization

To begin to address which mRNA metabolic processes might involve Slr1, we sought to determine the steady‐state subcellular localization of Slr1 by integrating a GFP tag at the 3′ end of SLR1. Wild type Slr1‐GFP expressed from its native promoter localizes predominantly to the nucleus of n class="Species">C. albicans, as detected by colocalization with DAPI (Fig. 3A, panel c). This steady state nuclear localization contrasts with the whole cell localization of SpSrp1 (Tang et al., 2007), but is similar to that seen for ScNpl3‐family proteins and the CBC (Shen et al., 2000; Yun and Fu, 2000; Gilbert et al., 2001; McBride et al., 2007; Tang et al., 2007). In addition, Slr1‐GFP appears in brighter puncta with slightly fainter fluorescence throughout the nucleus (Fig. 3B), reminiscent of the localization of metazoan SR proteins to nuclear speckles (Gui et al., 1994). This localization supports a model in which Slr1 has functions within the nucleus, but does not rule out cytoplasmic functions, as many SR‐like proteins are dynamic and shuttle between the nucleus and cytoplasm (Flach et al., 1994; Hacker and Krebber, 2004; Tang et al., 2007).

Figure 3

The C‐terminal arginine‐rich domain affects Slr1 nuclear localization.

A. Localization of WT and mutant Slr1‐GFP proteins. Cells expressing Slr1‐GFP were grown to mid‐log phase at 30°C and stained with DAPI in PBS prior to fluorescence microscopy. Wildtype Slr1‐GFP (panels a–d; SLR1‐GFP/slr1Δ) and slr1‐GFP proteins with deletions within the arginine‐rich domain from tyrosine 151 to glycine 192 (panels e–h; slr1Δ151‐192‐GFP/slr1Δ) or from aspartate 123 to arginine 194 (panels i–l; slr1Δ123‐194‐GFP/slr1Δ) were expressed from the native locus. Exposure times were equivalent for all strains and GFP images were merged with DAPI and DIC images in Adobe Photoshop CS5. Arrow = cell enlarged in B. Scale bar = 5 µm.

B. WT Slr1‐GFP is present in nuclear puncta. The cell indicated by an arrow in panels a–d of (A), is shown. The arrow indicates one region of brighter GFP fluorescence. Scale bar = 2 µm.

C. Quantification of subcellular localization. GFP, DAPI and DIC images of cells from the experiment in (A) were stacked in ImageJ (Schneider et al., 2012), the mean GFP fluorescence intensity and area of each cell (defined by DIC) and nucleus (defined by DAPI) were measured and used to calculate percent nuclear GFP fluorescence (%N) and the ratio of mean nuclear and mean cytoplasmic fluorescence intensity (N/C). Significant differences were detected among the cells of different genotypes (n = 100–110 per genotype; Kruskal–Wallis test, p < 0.0001). Significant differences by pairwise Mann–Whitney–Wilcoxon tests (*p < 0.0001) and the minimum, maximum, median and first and third quartiles are shown.

A. Localization of WT and mutant Slr1‐GFP proteins. Cells expressing Slr1‐GFP were grown to mid‐log phase at 30°C and stained with DAPI in n class="Chemical">PBS prior to fluorescence microscopy. Wildtype Slr1‐GFP (panels a–d; SLR1‐GFP/slr1Δ) and slr1‐GFP proteins with deletions within the arginine‐rich domain from tyrosine 151 to glycine 192 (panels e–h; slr1Δ151‐192‐GFP/slr1Δ) or from aspartate 123 to arginine 194 (panels i–l; slr1Δ123‐194‐GFP/slr1Δ) were expressed from the native locus. Exposure times were equivalent for all strains and GFP images were merged with DAPI and DIC images in Adobe Photoshop CS5. Arrow = cell enlarged in B. Scale bar = 5 µm.

C. Quantification of subcellular localization. GFP, DAPI and DIC images of cells from the experiment in (A) were stacked in ImageJ (Schneider et al., 2012), the mean GFP fluorescence intensity and area of each cell (defined by DIC) and nucn class="Chemical">leus (defined by DAPI) were measured and used to calculate percent nuclear GFP fluorescence (%N) and the ratio of mean nuclear and mean cytoplasmic fluorescence intensity (N/C). Significant differences were detected among the cells of different genotypes (n = 100–110 per genotype; Kruskal–Wallis test, p < 0.0001). Significant differences by pairwise Mann–Whitney–Wilcoxon tests (*p < 0.0001) and the minimum, maximum, median and first and third quartiles are shown.

The arginine‐rich domain of Scn class="Gene">Npl3 modulates its nucleocytoplasmic transport (Senger et al., 1998; McBride et al., 2005; Baierlein et al., 2013). To test whether the R‐rich domain influences the nuclear localization of Slr1, we deleted parts of this domain in Slr1‐GFP. Removal of 42 amino acids in the middle of this domain decreased the percentage of Slr1‐GFP found in the nucleus (%N) and the ratio of mean nuclear to mean cytoplasmic fluorescence intensity (N/C), indicating a slight shift to the cytoplasm at steady state (Fig. 3A, C, slr1Δ151‐192). Slr1 lacking an additional 30 residues primarily within the N‐terminal half of this domain, however, showed more distinct cytoplasmic localization than slr1Δ151‐192 (Fig. 3A, C; slr1Δ123‐194). These results were confirmed in two additional independent experiments. The increased detection of the mutant proteins in the cytoplasm did not result from protein instability: the mutant proteins were expressed at comparable levels to wildtype Slr1‐GFP (Supporting Information Fig. S2). In addition, the slight cytoplasmic localization of wildtype Slr1‐GFP was not due to autofluorescence: cells that did not express GFP had significantly lower mean cytoplasmic fluorescence than Slr1‐GFP‐expressing cells (Supporting Information Fig. S3). These results indicate the importance of the C‐terminal R‐rich domain in proper Slr1 localization.

Post‐translational modification of Slr1

Throughout eukaryotes, SR protein function and localization are modulated by phosphorylation at RS/n class="Chemical">SR dipeptides (Long and Caceres, 2009); in addition, arginine methylation within R‐rich domains influences subcellular localization and molecular interactions of many RNA‐binding proteins (Thandapani et al., 2013). The extensive R‐rich domain and the clustering of SR/RS dipeptides at the C‐terminus of Slr1 suggest that this region could be a target for modification.

To test for Slr1 phosphorylation, we constructed cells that expressed two different GFP‐tagged Slr1 proteins from the endogenous SLR1 promoter. In one set of strains, GFP was linked to the C‐terminus of wild type Slr1 (Slr1‐GFP); in the second set of strains, GFP integration was coupled with the introduction of mutations to substitute all six serines in C‐terminal n class="Chemical">SR/RS dipeptides with alanines (slr1‐6SA‐GFP; Fig. 1C, asterisks). These mutations did not affect log‐phase growth of C. albicans in rich medium at 30°C or 37°C (30°C generation time: WT = 198 ± 6 min (standard error of the mean SEM); 6SA = 203 ± 8 min SEM; 37°C generation time: WT = 182 ± 6 min SEM; 6SA = 190 ± 8 min SEM; n = 11, unpaired student's t‐test, p > 0.05). Anti‐GFP immunoblot analysis of Slr1‐GFP isolated from mid‐log phase yeast‐form cells revealed faster migration of the slr1‐6SA‐GFP mutant protein than the Slr1‐GFP wild type protein (Fig. 4A, compare lanes 1 and 4); the apparent difference (∼8 kDa) was much greater than would be expected based solely on the molecular weights of serine and alanine (difference <0.1 kDa) and could reflect phosphorylation of the wild type protein. The S‐to‐A mutations also led to an apparent increase in steady‐state levels of slr1‐6SA‐GFP compared with Slr1‐GFP (Fig. 4A, compare lanes 2 and 7).

Figure 4

Slr1 post‐translational modification.

A. Slr1 phosphorylation. Slr1‐GFP and slr1‐6SA‐GFP were immunoprecipitated from SLR1‐GFP/slr1Δ and slr1‐6SA‐GFP/slr1Δ yeast‐cell lysates and incubated with or without calf intestinal phosphatase for 1 h at 4°C or 37°C. Treated (+) and untreated (−) samples (precipitated from 1.6 mg or 0.32 mg total protein) were resolved by SDS‐PAGE and relative migration of Slr1 proteins detected by immunoblotting with anti‐GFP antibodies.

B. SR‐like proteins Npl3 and Slr1 are major targets for arginine methylation in C. albicans. Lysates from yeast cells expressing the major arginine methyltransferase Hmt1 with (HMT1/hmt1Δ) or without (HMT1/hmt1Δ npl3Δ/Δ) Npl3 were incubated with anti‐methylarginine antibody Ab412 (Abcam) and bound proteins purified with Protein G‐sepharose. Proteins were resolved by SDS‐PAGE and visualized by Coomassie staining. Cells lacking Hmt1 (hmt1Δ/Δ) were used to detect non‐specifically bound proteins.

C. Slr1‐GFP is arginine‐methylated. Cells expressing Slr1‐GFP with (+) and without (−) Hmt1 (SLR1‐GFP/slr1Δ and SLR1‐GFP/slr1Δ hmt1Δ/Δ) were lysed and Slr1‐GFP precipitated with anti‐GFP antibody. Methylation of Slr1‐GFP was detected by immunoblot with Ab412.

A. Slr1 phosphorylation. Slr1‐GFP and slr1‐6SA‐GFP were immunoprecipitated from SLR1‐GFP/slr1Δ and slr1‐6SA‐GFP/slr1Δ yeast‐cell lysates and incubated with or without n class="Species">calf intestinal phosphatase for 1 h at 4°C or 37°C. Treated (+) and untreated (−) samples (precipitated from 1.6 mg or 0.32 mg total protein) were resolved by SDS‐PAGE and relative migration of Slr1 proteins detected by immunoblotting with anti‐GFP antibodies.

B. SR‐like proteins n class="Gene">Npl3 and Slr1 are major targets for arginine methylation in C. albicans. Lysates from yeast cells expressing the major arginine methyltransferase Hmt1 with (HMT1/hmt1Δ) or without (HMT1/hmt1Δ npl3Δ/Δ) Npl3 were incubated with anti‐methylarginine antibody Ab412 (Abcam) and bound proteins purified with Protein G‐sepharose. Proteins were resolved by SDS‐PAGE and visualized by Coomassie staining. Cells lacking Hmt1 (hmt1Δ/Δ) were used to detect non‐specifically bound proteins.

C. Slr1‐GFP is arginine‐methylated. Cells expressing Slr1‐GFP with (+) and without (−) n class="Gene">Hmt1 (SLR1‐GFP/slr1Δ and SLR1‐GFP/slr1Δ hmt1Δ/Δ) were lysed and Slr1‐GFP precipitated with anti‐GFP antibody. Methylation of Slr1‐GFP was detected by immunoblot with Ab412.

To test phosphorylation directly, immunopurified GFP‐tagged proteins were incubated at 37°C with or without n class="Species">calf intestinal phosphatase. Phosphatase treatment of Slr1‐GFP resulted in two additional bands, one of which comigrated with untreated slr1‐6SA‐GFP (Fig. 4A, lanes 3 and 4), whereas no change in the migration of slr1‐6SA‐GFP was detected following phosphatase treatment (Fig. 4A, lanes 5 and 6). These results support a model where the phosphorylation of SR/RS dipeptides occurs in the C‐terminus of Slr1.

Purification of n class="Chemical">arginine‐methylated proteins from yeast‐form C. albicans cells also indicated that Slr1 is methylated (Fig. 4B). Anti‐methylarginine immunoprecipitation of proteins from wildtype C. albicans cell lysates revealed one major protein (Fig. 4B, lane 2) that was not seen in immunoprecipitates from cells lacking the major arginine methyltransferase, Hmt1 (lane 1). Mass spectrometric analysis identified this protein as CaNpl3, a known target of arginine methylation (McBride et al., 2007). Immunoprecipitation from npl3Δ/Δ lysates, however, increased detection of a second, slightly smaller arginine‐methylated protein, which was identified by mass spectrometry as Slr1 (Fig. 4B, lane 3). This modification of Slr1 was confirmed by immunoprecipitating Slr1‐GFP from strains with and without the methyltransferase and immunoblotting with the anti‐dimethylarginine antibody (Fig. 4C). The recognition of Slr1‐GFP by the anti‐methylarginine antibody in strains with Hmt1 indicated that its precipitation in Fig. 4B did not result from coprecipitation with another arginine‐methylated protein. Therefore, Slr1 is both phosphorylated and arginine‐methylated in C. albicans.

Modification of Slr1 influences its subcellular localization

Post‐translational modifications affect subcellular localization of a number of yeast n class="Chemical">SR proteins including SpSrp1 and ScNpl3‐family proteins ScNpl3, CaNpl3 and SpSrp2. All these RNA‐binding proteins move between the nucleus and the cytoplasm, but phosphorylation of ScNpl3‐family proteins facilitates their nuclear import (Yun and Fu, 2000; Gilbert et al., 2001; McBride et al., 2007; Tang et al., 2007), whereas phosphorylation of SpSrp1 enhances its nuclear export (Tang et al., 2007). In light of the sequence similarities between the N‐termini of Slr1 and SpSrp1 and the C‐termini of Slr1 and ScNpl3 proteins, we tested the impact of post‐translational modifications on Slr1 localization using mutational approaches.

Given the multiplicity of kinases that target SR proteins in other species (Yun and Fu, 2000; Gilbert et al., 2001; Tang et al., 2007; Dermody et al., 2008), to determine whether phosphorylation of Slr1 impacts its intracellular localization, localization of wiln class="Chemical">dtype Slr1‐GFP was compared with that of slr1‐6SA‐GFP. Introduction of the 6SA mutations caused a shift in the steady‐state localization: slr1‐6SA‐GFP is more cytoplasmic than wildtype Slr1‐GFP (Fig. 5A, compare panels a‐c and e‐g), with a significantly lower percent nuclear localization (%N) and ratio of mean nuclear to cytoplasmic fluorescence intensity (N/C) than WT Slr1‐GFP (Fig. 5D). These findings were confirmed in two additional independent experiments. The increased cytoplasmic localization of slr1‐6SA‐GFP was not due to the release of GFP from the fusion protein, as indicated by the absence of < 40 kDa proteins detected by an anti‐GFP antibody (Fig. 5E). In addition, slr1‐6SA‐GFP appeared in cytoplasmic foci, most notably at the bud neck of post‐mitotic cells (Fig. 5B and C). These results suggested that Slr1 phosphorylation may facilitate nuclear import, but may also have roles beyond regulation of Slr1 nucleocytoplasmic transport.

Figure 5

Serine mutation and arginine methylation affect Slr1 localization.

A. Localization of WT and mutant Slr1‐GFP proteins. Slr1‐GFP and slr1‐6SA‐GFP were expressed in cells with (SLR1‐GFP/slr1Δ, panels a–d; slr1‐6SA‐GFP/slr1Δ, panels e–h) or without (SLR1‐GFP/slr1Δ hmt1Δ/Δ, panels i–l; slr1‐6SA‐GFP/slr1Δ hmt1Δ/Δ, panels m–p) the arginine methyltransferase Hmt1 and visualized as in Fig. 3A. Exposure times were equivalent for all genotypes. Arrows indicate examples of post‐mitotic cells with slr1‐6SA‐GFP foci at the bud neck. Scale bar = 5 µm.

B. Bud‐neck localization of slr1‐6SA‐GFP in post‐mitotic cells with Hmt1. Examples of cells with 1 (arrow, panels a, c, e), 2 (arrows, panels b, d, f) or no (asterisk, panels b, d, f) bud‐neck slr1‐6SA‐GFP focus from panels h, g and e in Fig. 5A are shown. Scale bar = 5 µm.

C. Bud‐neck localization of slr1‐6SA‐GFP in post‐mitotic cells without Hmt1. Examples of cells from panels p, o and m in Fig. 5A are shown (arrow = bud‐neck focus). Scale bar = 5 µm.

D. Quantification of subcellular localization. GFP, DAPI and DIC images of cells from the experiment in Fig. 5A were analyzed as in Fig. 3C to determine percent nuclear GFP fluorescence (%N) and the ratio between the mean nuclear and mean cytoplasmic GFP fluorescence intensity (N/C). Significant differences were detected among the cells of different genotypes (n = 59–66 for each genotype; Kruskal–Wallis test, p < 0.0001). Significant differences by pairwise Mann–Whitney–Wilcoxon tests (*p < 0.0001) and the minimum, maximum, median and first and third quartiles are shown. WT = wildtype Slr1‐GFP, 6SA = slr1‐6SA‐GFP.

E. Absence of arginine methylation does not decrease Slr1 protein levels. Cells in Fig. 5A were grown to mid‐log phase in YPD, lysed and levels of GFP proteins in 17 µg total protein analyzed by SDS‐10% PAGE and anti‐GFP immunoblot.

Serine mutation and n class="Chemical">arginine methylation affect Slr1 localization.

A. Localization of WT and mutant Slr1‐GFP proteins. Slr1‐GFP and slr1‐6SA‐GFP were expressed in cells with (SLR1‐GFP/slr1Δ, panels a–d; slr1‐6SA‐GFP/slr1Δ, panels e–h) or without (SLR1‐GFP/slr1Δ hmt1Δ/Δ, panels i–l; slr1‐6SA‐GFP/slr1Δ n class="Gene">hmt1Δ/Δ, panels m–p) the arginine methyltransferase Hmt1 and visualized as in Fig. 3A. Exposure times were equivalent for all genotypes. Arrows indicate examples of post‐mitotic cells with slr1‐6SA‐GFP foci at the bud neck. Scale bar = 5 µm.

D. Quantification of subcellular localization. GFP, DAPI and DIC images of cells from the experiment in Fig. 5A were analyzed as in Fig. 3C to determine percent nuclear GFP fluorescence (%N) and the ratio between the mean nuclear and mean cytoplasmic GFP fluorescence intensity (N/C). Significant differences were detected among the cells of different genotypes (n = 59–66 for each genotype; Kruskal–Wallis test, p < 0.0001). Significant differences by pairwise Mann–Whitney–Wilcoxon tests (*p < 0.0001) and the minimum, maximum, median and first and third quartiles are shown. WT = wiln class="Chemical">dtype Slr1‐GFP, 6SA = slr1‐6SA‐GFP.

E. Absence of arginine methylation does not decrease Slr1 protein levels. Cells in Fig. 5A were grown to mid‐log phase in n class="Chemical">YPD, lysed and levels of GFP proteins in 17 µg total protein analyzed by SDS‐10% PAGE and anti‐GFP immunoblot.

Arginine methylation facilitates nuclear export of both Scn class="Gene">Npl3 and CaNpl3 (Shen et al., 1998; McBride et al., 2007). To test whether methylation affects nucleocytoplasmic transport of Slr1, Slr1‐GFP was expressed in cells lacking the methyltransferase Hmt1 (Fig. 5). The predominantly nuclear localization of Slr1‐GFP in the absence of Hmt1 indicates that methylation is not required for nuclear import (Fig. 5A, panels i–k). No significant difference in the %N and N/C ratio of WT Slr1‐GFP was detected between cells with and without Hmt1 (Fig. 5A, compare panels a–c and i–k; Fig. 5D). Both measures of nuclear slr1‐6SA‐GFP localization, however, were significantly increased by deletion of HMT1 (Fig. 5D). This subtle increase in nuclear slr1‐6SA‐GFP in the absence of Hmt1 was confirmed in two additional independent experiments (N/C) and one of two additional experiments (%N). The apparent decrease in cytoplasmic slr1‐6SA‐GFP was not due to lower levels of expression of slr1‐6SA‐GFP in the absence of Hmt1 (Fig. 5E, compare lanes 2 and 4). The percentage of post‐mitotic cells with slr1‐6SA‐GFP foci at the bud neck, however, was not significantly different in the presence (53% ± 8%SEM) and absence (47% ± 1%SEM) of arginine methylation (> 55 cells per strain in three independent experiments; p > 0.05, student's t‐test). Thus, the R‐rich domain, arginine methylation and phosphorylation of Slr1 affect its intracellular localization.

Slr1 is not required for bulk mRNA export

The binding of Slr1 to mRNA in a complex with the CBC, combined with its ability to move between the nucleus and cytoplasm, suggested that Slr1, like Scn class="Gene">Npl3, might play a role in nuclear export of mRNA (Singleton et al., 1995; Lee et al., 1996; Shen et al., 2000). We, therefore, developed a fluorescence in situ hybridization (FISH) assay with an oligo(dT) probe to detect the localization of bulk mRNA in cells with and without Slr1 (Fig. 6). In S. cerevisiae, such assays detect the cytoplasmic localization of polyadenylated mRNA, which becomes predominantly nuclear in the presence of mutations that block mRNA export (Singleton et al., 1995). In addition, heat shock treatment of S. cerevisiae at 42°C blocks bulk mRNA export while not affecting export of heat shock mRNAs (Saavedra et al., 1996; Tani et al., 1996).

Figure 6

SLR1 deletion does not block bulk mRNA export.

Cells with (SLR1/SLR1; AMC79) and without (slr1Δ/slr1Δ; AMC89) Slr1 were grown at 30°C, 37°C, or shifted to 42°C for 30 min. after growth at 30°C before fixation. Fixed cells were hybridized with digoxigenin‐conjugated oligo(dT), and stained with DAPI and FITC‐conjugated anti‐digoxigenin antibody. Nuclei and bulk poly(A) mRNAs were visualized by fluorescence microscopy using the DAPI and GFP filters respectively. Exposure times were equivalent for all strains and growth conditions. Scale bar = 5 µm.

Cells with (SLR1/SLR1; AMC79) and without (slr1Δ/slr1Δ; AMC89) Slr1 were grown at 30°C, 37°C, or shifted to 42°C for 30 min. after growth at 30°C before fixation. Fixed cells were hybridized with digoxigenin‐conjugated n class="Chemical">oligo(dT), and stained with DAPI and FITC‐conjugated anti‐digoxigenin antibody. Nuclei and bulk poly(A) mRNAs were visualized by fluorescence microscopy using the DAPI and GFP filters respectively. Exposure times were equivalent for all strains and growth conditions. Scale bar = 5 µm.

To ascertain that our assay could detect a block to nuclear mRNA export, we tested whether heat shock treatment at 42°C also resulted in nuclear localization of bulk poly(A) RNA in n class="Species">C. albicans (Fig. 6). Polyadenylated RNA was found throughout C. albicans cells at 30°C and 37°C (Fig. 6, panels B, F), but accumulated in the nucleus at 42°C (panel J). In the absence of Slr1, however, no nuclear accumulation of bulk poly(A) RNA was detected (Fig. 6, panels D, H) except during heat‐shock conditions (panel L). Therefore, although Slr1 moves between nuclear and cytoplasmic compartments, it is dispensable for nuclear export of the majority of polyadenylated mRNAs under standard conditions.

Slr1 co‐fractionates with translating ribosomes

The detection of a fraction of wildtype Slr1‐GFP outside the n class="Chemical">DAPI‐stained nucleus (Fig. 5D), in addition to the cytoplasmic localization of slr1‐6SA‐GFP, suggested that Slr1 might function in part in the cytoplasm. S. cerevisiae SR‐like protein Npl3 is required for monosome formation during translation initiation and co‐fractionates with polysomes (Windgassen et al., 2004; Baierlein et al., 2013). We, therefore, compared the migration of wildtype Slr1‐GFP with that of ribosomal protein Rps3 through sucrose density gradients (Fig. 7). While the majority of Slr1‐GFP was detected in non‐ribosomal and ribosomal subunit fractions (79%; Fig. 7B), 21% was found in fractions that contained the 80S ribosome and polysomes. This association of Slr1‐GFP with 80S and translating ribosomes was abrogated by treatment of lysates with RNase A prior to centrifugation (Fig. 7C). These results indicated that wildtype Slr1 is present in the cytoplasm and suggested that wildtype Slr1 may play a role in translation through binding to mRNA (Fig. 2A).

Figure 7

Slr1‐GFP co‐fractionates with 80S and translating ribosomes.

A. Log‐phase yeast cells expressing Slr1‐GFP (SLR1‐GFP/slr1Δ) were treated with cycloheximide prior to lysis. Lysates were loaded on linear 7%–47% sucrose gradients and fractionated following centrifugation. RNA absorbance at 254 nm was measured during fractionation to detect 40S and 60S ribosomal subunits, 80S ribosomes and polysomes.

B. TCA‐precipitated fractions were resolved by SDS‐10% PAGE and tested for the presence of Slr1‐GFP and ribosomal protein Rps3 by immunoblot (GFP, uS3). The percent of each protein present in non‐ribosomal and ribosomal fractions was determined using Image StudioLite software (LI‐COR).

C. Lysates were treated with RNase A prior to sucrose‐density gradient centrifugation and then analyzed as in (B).

A. Log‐phase yeast cells expressing Slr1‐GFP (SLR1‐GFP/slr1Δ) were treated with n class="Chemical">cycloheximide prior to lysis. Lysates were loaded on linear 7%–47% sucrose gradients and fractionated following centrifugation. RNA absorbance at 254 nm was measured during fractionation to detect 40S and 60S ribosomal subunits, 80S ribosomes and polysomes.

B. TCA‐precipitated fractions were resolved by n class="Chemical">SDS‐10% PAGE and tested for the presence of Slr1‐GFP and ribosomal protein Rps3 by immunoblot (GFP, uS3). The percent of each protein present in non‐ribosomal and ribosomal fractions was determined using Image StudioLite software (LI‐COR).

slr1‐6SA localizes to the hyphal tip

The deletion of SLR1 decreases hyphal growth and function (Ariyachet et al., 2013), raising the question of whether Slr1 is present in hyphal cells. When cells were induced to form hyphae, wiln class="Chemical">dtype Slr1‐GFP localized to both mother cell and hyphal nuclei (Fig. 8A, panels c–e). Interestingly, slr1‐6SA‐GFP appeared not only in the nuclei and cytoplasm, but also in cytoplasmic foci along the hypha, including an intense spot near the tip of the hypha (Fig. 8B, panel c–e, arrow). In addition, slr1‐6SA‐GFP accumulated at the tip of hyphal branches (Fig. 8C, arrowhead), another region of polarized growth, and at some septa (Fig. 8D, arrowhead), similar to its appearance at the bud neck of yeast‐form cells (Fig. 5B). The predominant slr1‐6SA‐GFP hyphal tip focus was reminiscent of the Spitzenkörper, a structure found near hyphal tips of filamentous fungi (Riquelme, 2013). The vesicles that comprise the Spitzenkörper deliver protein and lipid cargoes to the growing hyphal tip (Riquelme, 2013).

Figure 8

Serine mutations shift localization of Slr1‐GFP to hyphal foci.

A. Cells expressing wildtype Slr1‐GFP (SLR1‐GFP/slr1Δ) were grown in RPMI at 37°C to induce hyphal formation. After 3 h, cells were stained with DAPI to visualize nuclei and localization of Slr1‐GFP proteins was determined by fluorescence microscopy using GFP and DAPI filters. Arrowhead = mother cell nucleus; arrow = hyphal nucleus.

B. Cells expressing slr1‐6SA‐GFP (slr1‐6SA‐GFP/slr1Δ) were grown and imaged as in (A). Arrow = hyphal tip focus.

C. Cells expressing slr1‐6SA‐GFP were grown and imaged as in (B). Arrowhead = hyphal branch focus.

D. Cells expressing slr1‐6SA‐GFP were grown and imaged as in (B). Arrowhead = septal focus. Exposure times were equivalent for both genotypes. Scale bar = 10 µm.

Serine mutations shift localization of Slr1‐GFP to n class="Disease">hyphal foci.

A. Cells expressing wildtype Slr1‐GFP (SLR1‐GFP/slr1Δ) were grown in n class="Chemical">RPMI at 37°C to induce hyphal formation. After 3 h, cells were stained with DAPI to visualize nuclei and localization of Slr1‐GFP proteins was determined by fluorescence microscopy using GFP and DAPI filters. Arrowhead = mother cell nucleus; arrow = hyphal nucleus.

We therefore tested for colocalization of the hyphal tip slr1‐6SA‐GFP focus and the Spitzenkörper (Fig. 9, Table 1). Cells incorporate the lipophilic dye FM4‐64 into endocytic vesicles, leading to rapid accumulation in vacuolar membranes (Vida and Emr, 1995). In growing hyphae, however, FM4‐64 can be seen in the Spitzenkörper in a brief window after exposure to the dye (Crampin et al., 2005). n class="Species">Yeast‐form cells expressing slr1‐6SA‐GFP were diluted in hyphal‐inducing medium, incubated at 37°C and exposed to FM4‐64 three hours after hyphal induction. In 65% of cells with distinct Spitzenkörper FM4‐64 staining, this fluorescence partially colocalized with a slr1‐6SA‐GFP focus at the hyphal tip (Fig. 9A, panels b–d) and in 4%–8% of hyphae, an slr1‐6SA‐GFP focus was adjacent to the Spitzenkörper (Table 1). The ability of FM4‐64 to stain the Spitzenkörper was confirmed by hyphal tip colocalization of FM4‐64 with a fluorescent myosin light‐chain 1 fusion protein (Mlc1‐YFP), a known Spitzenkörper protein (Crampin et al., 2005) (Fig. 9B, Table 1). Line scans demonstrating the partial colocalization of slr1‐6SA‐GFP and Mlc1‐YFP with the Spitzenkörper are shown in Supporting Information Fig. S4. The localization of slr1‐6SA‐GFP to a region of the hypha near the Spitzenkörper suggested that this protein might associate with transport complexes, for example by binding to mRNAs that are transported to the hyphal tip.

Figure 9

The slr1‐6SA‐GFP hyphal tip focus partially overlaps with the Spitzenkörper.

A. Cells expressing slr1‐6SA‐GFP (SLR1‐GFP/slr1Δ) were induced to form hyphae as in Fig. 8. After 3 h, FM4‐64 was added and cells incubated at 37°C for 4 min. Cells were quickly washed with warm PBS and visualized by fluorescence microscopy with Texas Red (FM4‐64) and GFP (slr1‐6SA‐GFP, Mlc1‐YFP) filters. Arrows in panels a–e indicate hyphal tips enlarged in panels f–j. Scale bars: panels a–e = 5 µm, panels f–j = 1 µm.

B. Cells expressing Mlc1‐YFP (MLC1/MLC1‐YFP) were grown, stained, visualized and labeled as in (A).

Table 1

Partial overlap of slr1‐6SA‐GFP hyphal tip foci with the Spitzenkörper.

	slr1‐6SA‐GFP		Mlc1‐YFP
Experiment a	A	B	A	B
GFP/YFP focus b
Partial overlap with Spitzenkörper	65% (43)	65% (15)	98% (63)	95% (21)
Adjacent to Spitzenkörper	8% (5)	4% (1)	0% (0)	0% (0)
No GFP/YFP focus at hyphal tip	27% (18)	30% (7)	2% (1)	5% (1)
Total # hyphae scored	66	23	64	22

a. FM4‐64 staining of the Spitzenkörper was compared with slr1‐6SA‐GFP and Mlc1‐YFP hyphal tip fluorescence in two experiments (A, B).

b. GFP/YFP foci for all hyphae with distinct FM4‐64 staining of the Spitzenkörper were scored as partially overlapping with or adjacent to the Spitzenkörper, or absent from the hyphal tip. The absolute number of hyphae in each category is noted in parentheses.

A. Cells expressing slr1‐6SA‐GFP (SLR1‐GFP/slr1Δ) were induced to form hyphae as in Fig. 8. After 3 h, FM4‐64 was added and cells incubated at 37°C for 4 min. Cells were quickly washed with warm PBS and visualized by fluorescence microscopy with n class="Chemical">Texas Red (FM4‐64) and GFP (slr1‐6SA‐GFP, Mlc1‐YFP) filters. Arrows in panels a–e indicate hyphal tips enlarged in panels f–j. Scale bars: panels a–e = 5 µm, panels f–j = 1 µm.

B. Cells expressing Mlc1‐YFP (n class="Gene">MLC1/MLC1‐YFP) were grown, stained, visualized and labeled as in (A).

a. FM4‐64 staining of the Spitzenkörper was compared with slr1‐6SA‐GFP and Mlc1‐YFP n class="Disease">hyphal tip fluorescence in two experiments (A, B).

In S. cerevisiae, the n class="Gene">She3 protein complex transports a set of mRNAs to the bud tip (Shepard et al., 2003). The C. albicans She3 ortholog binds to at least 37 mRNAs during hyphal growth (Elson et al., 2009). At least 12 of these transcripts localize to the hyphal tip and this localization requires the CaShe3 protein (Elson et al., 2009). We hypothesized that the accumulation of the mRNA‐binding protein slr1‐6SA at the hyphal tip might depend on mRNA transport to the tip. We therefore expressed slr1‐6SA‐GFP in C. albicans strains without CaShe3 (Fig. 10). In cells lacking She3, many fewer cells contained slr1‐6SA‐GFP foci at the hyphal tip (Fig. 10A, panels g–l); the percentage of hyphal tips containing slr1‐6SA‐GFP foci is over three times lower in strains without She3 than in strains bearing a single copy of SHE3 (Fig. 10B). In contrast, slr1‐6SA‐GFP formed cytoplasmic foci in the presence or absence of She3 (Fig. 10A). The average percentage of hyphal tips with slr1‐6SA‐GFP foci in the presence of She3 (70.3% ± 2.6%SEM) was consistent with the average percentage of hyphae with slr1‐6SA‐GFP foci that partially overlapped with (65%) or were adjacent to (4%–8%) the Spitzenkörper (Table 1). These results indicated that slr1‐6SA‐GFP localization specifically to the hyphal tip is partially dependent on She3, suggesting the association of slr1‐6SA‐GFP with She3‐transported mRNAs.

Figure 10

Absence of RNA‐binding protein She3 decreases slr1‐6SA‐GFP hyphal tip localization.

A. Deletion of SHE3 decreases hyphal tip localization of slr1‐6SA‐GFP. Cells expressing slr1‐6SA‐GFP in the presence (slr1‐6SA‐GFP/SLR1 SHE3/she3Δ) and absence (slr1‐6SA‐GFP/SLR1 she3Δ/she3Δ) of She3 were grown and visualized as in Fig. 8. Arrows indicate the most distal slr1‐6SA‐GFP focus in each hypha. Scale bar = 5 µm.

B. Quantification of slr1‐6SA‐GFP hyphal tip localization. Cells containing distal GFP foci either within 2 µm of the hyphal tip or farther than 2 µm from the hyphal tip were counted (≥ 65 cells per strain). Percentages of cells with slr1‐6SA‐GFP at the hyphal tip were significantly different between the two strains (*p < 0.0001; student's t‐test). Mean values from three independent trials and standard error of the mean are shown.

Absence of RNA‐binding protein She3 decreases slr1‐6SA‐GFP n class="Disease">hyphal tip localization.

A. Deletion of SHE3 n class="Disease">decreases hyphal tip localization of slr1‐6SA‐GFP. Cells expressing slr1‐6SA‐GFP in the presence (slr1‐6SA‐GFP/SLR1 SHE3/she3Δ) and absence (slr1‐6SA‐GFP/SLR1 she3Δ/she3Δ) of She3 were grown and visualized as in Fig. 8. Arrows indicate the most distal slr1‐6SA‐GFP focus in each hypha. Scale bar = 5 µm.

B. Quantification of slr1‐6SA‐GFP hyphal tip localization. Cells containing distal GFP foci either within 2 µm of the n class="Disease">hyphal tip or farther than 2 µm from the hyphal tip were counted (≥ 65 cells per strain). Percentages of cells with slr1‐6SA‐GFP at the hyphal tip were significantly different between the two strains (*p < 0.0001; student's t‐test). Mean values from three independent trials and standard error of the mean are shown.

Discussion

Post‐transcriptional regulation of gene expression has been linked to cellular differentiation throughout eukaryotes. The absence of putative RNA‐binding protein Slr1 delays hyphal formation in C. albicans, lowers virulence and alters exposure of a hyphal cell‐wall‐associated protein involved in host–cell interactions, suggesting the involvement of Slr1 in post‐transcriptional processes that influence hyphal formation and function (Ariyachet et al., 2013). We now demonstrate that Slr1 is a component of an mRNP complex and is found in both the nucn class="Chemical">leus and the cytoplasm. Slr1 shares primary structural features with two different fungal SR‐like RNA‐binding protein families: the Npl3/Srp2 proteins, which are essential in S. cerevisiae and S. pombe, and SpSrp1‐like proteins, which have not previously been identified in the Saccharomycetale lineage (Plass et al., 2008).

Fungal SR‐like protein divergence

Comparison of the primary sequence of Slr1 to other fungal proteins revealed that whereas the C‐terminal arginine‐rich domain is most similar to Ascomycete n class="Gene">Npl3‐like proteins, the N‐terminal RNA‐recognition motif is homologous to that of SpSrp1 (Fig. 1). BLAST searches with the single RRM of SpSrp1 and Slr1 revealed the wider presence of potentially related proteins than initially suggested (Plass et al., 2008; Supporting Information Fig. S1). The absence of the identification of these putative SpSrp1 homologs in previous work may be due in part to the divergence within a peptide found in SR proteins that overlaps with RNP‐1 (Fig. 1B; Supporting Information Fig. S1; Birney et al., 1993). The conservation of the 5′ splice site among the intron‐containing SRP1 homologs (after the second nucleotide of the codon for the underlined residue in Fig. 1B), however, supports an evolutionary relationship among these genes.

SR‐like protein modification and localization

Although the predominance of RG dipeptides in the Slr1 n class="Chemical">arginine‐rich C‐terminal domain more closely resembles ScNpl3 than SpSrp1, the clustering of SR/RS dipeptides in C‐terminal 20 amino acids of Slr1 (Fig. 1C) is more similar to SpSrp1 and many metazoan SR proteins (Wilson‐Grady et al., 2008). This clustering of SR/RS dipeptides in Slr1 raises the question of whether the regulation of Slr1 by phosphorylation may be more similar to that of SpSrp1 family proteins than that of ScNpl3. Phosphatase treatment of Slr1‐GFP and slr1‐6SA‐GFP indicated that Slr1 is likely phosphorylated at multiple sites within the SR region (Fig. 4A). Phosphorylation of one or more residues may control additional phosphorylation events, as seen in the sequential phosphorylation of serine residues in human SR protein ASF/SF2 (Ngo et al., 2008). Whereas wildtype Slr1‐GFP is predominantly nuclear, the S‐to‐A substitutions increase cytoplasmic levels of Slr1‐GFP, suggesting that phosphorylation promotes import of Slr1 (Fig. 5), potentially by facilitating interaction with a conserved Mtr10 import receptor complex, as seen for S. cerevisiae Npl3 (Yun and Fu, 2000). Phosphorylation of the SR domain of multiple metazoan SR proteins similarly allows binding to the importin transportin‐SR (Lai et al., 2000). The cytoplasmic localization of slr1‐6SA‐GFP in C. albicans decreases in the absence of arginine methyltransferase Hmt1 (Fig. 5), supporting a role for methylation in nuclear export of Slr1, as seen for ScNpl3 and CaNpl3 (Shen et al., 1998; McBride et al., 2007). Therefore, the impact of post‐translational modification on Slr1 nucleocytoplasmic transport is more similar to the effects of modifications on Npl3 family proteins than on SpSrp1, correlating with greater similarity of the Npl3 and Slr1 R‐rich domains rather than with the clustered arrangement of SR/RS motifs.

Interestingly, subcellular localization of slr1‐6SA‐GFP was also detected in cytoplasmic foci in both budding and hyphal cells, including near the bud neck and hyphal tip. These foci could reflect increased interaction of cytoplasmic unphosphorylated slr1‐6SA‐GFP with ribonucleoprotein complexes in subcellular domains such as RNA‐processing bodies (P‐bodies), stress granules (Buchan and Parker, 2009), or target regions for mRNA transport. P‐bodies accumulate dn class="Chemical">uring C. albicans hyphal induction (Jung and Kim, 2011), but they are not specifically detected at the hyphal tip; the less intense slr1‐6SA‐GFP foci present throughout the hypha (e.g., Fig. 10A) may indicate association of slr1‐6SA‐GFP with these RNA‐rich cytoplasmic regions. Lack of phosphorylation of an SR motif in the severe acute respiratory syndrome coronavirus (SARS CoV) nucleocapsid protein has been implicated in accumulation of this viral SR protein in stress granules (Peng et al., 2008). The localization of slr1‐6SA‐GFP to cytoplasmic foci suggests that phosphorylation may prevent accumulation of this SR‐like protein in similar mRNA‐rich structures.

We hypothesize that Slr1 may aid in transport of mRNAs in C. albicans and that its phosphorylation may promote its dissociation from mRNP complexes in distinct cytoplasmic sites such as the bud neck and the n class="Disease">hyphal tip. In S. cerevisiae, the absence of the kinase ScSky1 or the presence of an S‐to‐A mutation in ScNpl3 increases the binding of ScNpl3 to poly(A) RNA, indicating a role for phosphorylation in promoting cytoplasmic mRNA release as well as in nuclear ScNpl3 import (Gilbert et al., 2001). CaShe3 is the primary C. albicans protein known to facilitate mRNA transport to the hypha (Elson et al., 2009); this fungus lacks an ortholog of ScShe2, a key mRNA‐binding protein in S. cerevisiae that couples nuclear export of ASH1 mRNA to formation of the She3 mRNA transport complex in the cytoplasm (Bohl et al., 2000). While slr1‐6SA‐GFP still forms cytoplasmic foci in hyphal cells lacking CaShe3, the absence of CaShe3 reduces the number of cells with foci at the hyphal tip (Fig. 10). This result suggests that slr1‐6SA‐GFP may travel with CaShe3 mRNA transport complexes to the hyphal tip.

The localization of the slr1‐6SA‐GFP focus at the hyphal tip is remarkably similar to that of the Spitzenkörper, a vesicular structure found in n class="Disease">filamentous fungi thought to aid in transport of proteins and lipids to and from the hyphal tip (Riquelme, 2013). FM4‐64 staining of the Spitzenkörper in strains expressing slr1‐6SA‐GFP indicated that these two structures partially overlap (Fig. 9A; Supporting Information Fig. S4). Sudbery and colleagues recently demonstrated that the C. albicans Sec2 protein, a Guanine Exchange Factor involved in vesicular transport to the hyphal tip, binds to the Sec2 mRNA and that wildtype Sec2 mRNA and protein colocalize in the Spitzenkörper (Caballero‐Lima et al., 2014). A phosphomimetic mutation in Sec2 decreases Sec2 protein binding to and colocalization with Sec2 mRNA in the Spitzenkörper (Caballero‐Lima et al., 2014). These results support a model in which phosphorylation of Sec2 helps regulate Sec2 mRNA transport to the hyphal tip (Caballero‐Lima et al., 2014). Phosphorylation of She3, Khd1 and Puf6 RNA‐binding proteins in S. cerevisiae has also been linked to polarized ASH1 mRNA transport and translation regulation (Paquin et al., 2007; Deng et al., 2008; Landers et al., 2009). The colocalization of slr1‐6SA‐GFP with the Spitzenkörper suggests both that mRNA‐binding proteins in addition to Sec2 and She3 could influence transport of mRNAs to the hyphal tip and that such mRNP complexes could be regulated similarly by phosphorylation of RNA‐binding proteins including Slr1.

SR‐like protein function

SR proteins have been implicated in many steps of mRNA metabolism, from linking transcription to splicing, controlling constitutive and alternative splicing, and directing mRNA nuclear export to affecting mRNA turnover and translation in the cytoplasm (Long and Caceres, 2009; Shepard and Hertel, 2009; Zhong et al., 2009). The RNA‐dependent interaction of Slr1 with the nuclear mRNA cap‐binding complex, which affects mRNA splicing, transport, stability and translation in n class="Species">S. cerevisiae (Topisirovic et al., 2011; Garre et al., 2012), suggests that Slr1 may also have complex roles in Candida RNA metabolism. The cytoplasmic localization of bulk mRNA in slr1Δ/Δ cells indicates that Slr1 is not crucial for bulk mRNA transport (Fig. 6), yet the co‐fractionation of wildtype Slr1‐GFP with 80S and translating ribosomes on sucrose‐density gradients (Fig. 7) suggests that Slr1 may have a cytoplasmic role in translation. In addition, the localization of slr1‐6SA‐GFP raises the question whether Slr1 might also function in mRNA transport to the bud neck (Fig. 5) or hyphal tip (Figs. 8, 9, 10).

Although deletion of SLR1 in C. albicans, like the C‐terminal truncation of its ortholog SwoK in n class="Species">Aspergillus nidulans, causes defects in polarized growth of these filamenting fungi (Shaw and Upadhyay, 2005; Ariyachet et al., 2013), the six S‐to‐A substitutions in Slr1 do not disrupt its critical functions. Yeast cells expressing mutant and wildtype Slr1‐GFP proteins have similar growth rates and the slr1‐6SA‐GFP mutant cells can form hyphae. These results, combined with the presence of low levels of slr1‐6SA‐GFP in hyphal nuclei, are consistent with a model in which Slr1 shuttles between the nucleus and the hyphal tip and Slr1 phosphorylation facilitates, but is not absolutely required for, release of mRNAs at the hyphal tip.

In conclusion, the unphosphorylated slr1‐6SA protein is the first C. albicans protein with a defined RNA‐binding domain to be found at the n class="Disease">hyphal tip and this localization depends on the known mRNA transport protein CaShe3. The importance of Slr1 for hyphal formation and function may therefore be due in part to Slr1 having a role in hyphal mRNA transport. For example, in the absence of a C. albicans She2 ortholog, Slr1 might link export of mRNAs required at the hyphal tip to cytoplasmic She3 mRNP formation. The slr1‐6SA mutant protein will serve as a particularly useful tool for future studies to identify the protein and mRNA components of ribonucleoprotein complexes that could impact hyphal growth and function.

Experimental procedures

C. albicans strains and growth conditions

Genotypes and important features of the strains, plasmids and oligonucleotides used in this study are described in Supporting Information Tables S1, S2 and S3 respectively. Most n class="Species">C. albicans strains in this study were derived from the arginine‐, histidine‐, uridine‐auxotrophic strain BWP17 (Wilson et al., 1999). The other uridine‐auxotrophic parental strains with SHE3 deletions are described in (Elson et al., 2009). All oligonucleotides were synthesized at Integrated DNA Technologies. Strain construction is described in detail in Supporting Information.

C. albicans strains were grown in n class="Chemical">YPD medium (1% yeast extract, 2% bactopeptone, 2% glucose) supplemented with 80 µg ml−1 uridine (Uri) or in synthetic dropout media with 2% glucose and lacking the appropriate nutrients to select for integrated markers. Generation times for strains expressing wildtype SLR1‐GFP or slr1‐6SA‐GFP as the sole copy of SLR1 (11 replicate cultures per strain) were calculated from 24 h growth curves as described in (Ariyachet et al., 2013). For optimal filamentation in broth cultures, strains were grown overnight in YPD + Uri, diluted to 3 × 106 cells ml−1 into pre‐warmed HEPES‐buffered RPMI 1640 (Life Technologies) and incubated at 37°C with shaking for 3 h.

SR‐like protein sequence analysis

A BLASTP search with amino acids 5–51 of Slr1, representing the core of the RNA‐recognition motif from RNP‐2 through RNP‐1 (Birney et al., 1993), identified proteins similar to Slr1 in many fungi. The following sequences were used to align the RRMs of twelve of these representative fungal SR‐like proteins: XP_002553256.1 (n class="Species">L. thermotolerans), XP_452519.1 (K. lactis), NP_985205.1 (A. gossypii), XP_500797.1 (Y. lipolytica), CAA22007.1 (C. albicans), XP_459563.2 (D. hansenii), XP_956575.2 (N. crassa), XP_663406.1 (A. nidulans), NP_596398.1 (S. pombe Srp1), XP_758616.1 (U. maydis) and XP_001731715.1 (M. globosa). A BLASTP search of the L. kluyveri genome also revealed a similar protein encoded by SAKL0H05192 (http://www.genolevures.org/blast.html). RNA‐recognition motifs were identified using an InterProScan sequence search (HMMPfam, PF00076; http://www.ebi.ac.uk/Tools/pfa/iprscan/; Zdobnov and Apweiler, 2001) and aligned using Clustalw2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/; Larkin et al., 2007; Goujon et al., 2010). Given the shorter length of other predicted Slr1‐like RRMs, up to 5 residues were removed from the C‐terminus of the predicted RRM domains of M. globosa, U. maydis, A. nidulans, N. crassa and K. lactis Slr1‐like proteins in the alignments shown in Fig. 1B and Supporting Information Fig. S1.

Testing Slr1 R‐rich domain function in S. cerevisiae Npl3

To study the functional similarity between ScNpl3 and Slr1, the R‐rich domain (codons I279‐R402) of Scn class="Gene">Npl3 with or without the C‐terminal heptapeptide (codons T403‐R414) was replaced with that of Slr1. To take advantage of the high level of homologous recombination in S. cerevisiae to construct these hybrid plasmids, the coding region for the R‐rich domain of Slr1 was amplified from BWP17 genomic DNA using AM322/AM323 (codons Q90‐G233, lacking the Slr1 C‐terminus) or oligos AM322/AM324 (codons Q90‐Y263, including the Slr1 C‐terminus). Each fragment was co‐transformed into wildtype S. cerevisiae strain FY23 with ApaI/NsiI‐cut pAM463 (PrA‐ScNPL3‐ApaI) (McBride et al., 2007) and plasmids were rescued from Leu+ cells. Resulting plasmids pAM471 (encoding ScNpl3 with the Slr1 R‐rich domain and the ScNpl3 C‐terminus) and pAM472 (encoding ScNpl3 with the Slr1 R‐rich domain and Slr1 C‐terminus) were sequenced at Geneway Research to ensure proper fusion.

To monitor the ability of the Slr1 C‐terminus to support S. cerevisiae n class="Gene">Npl3 function, plasmids encoding chimeric ScNpl3 proteins fused to Protein A, or the PrA LEU2 vector pNOPPATA (Hellmuth et al., 1998), were transformed into an S. cerevisiae npl3Δ strain bearing a wildtype NPL3 URA3 plasmid (PSY814; npl3Δ::HIS3 ade2 ade8 can1 ura3 leu2 his3 lys1 trp1 + YCp50‐NPL3‐3 MATa (Henry et al., 1996)). The ability of transformed cells to lose the ScNPL3 plasmid was detected on plates containing 5‐fluoro‐orotic acid as previously described (McBride et al., 2007).

Cell lysis for protein expression and purification

C. albicans cells were grown to mid‐log phase at 30°C. Cells were harvested and resuspended in minimal lysis buffer. All lysis buffers contained protease inhibitors: n class="Chemical">phenylmethylsulfonyl fluoride (1 mM), pepstatin, leupeptin, aprotinin, antipain and chymostatin (2.5 μg ml−1 each). Lysis buffers included: RIPA buffer (radio immunoprecipitation assay buffer: 50 mM Tris‐HCl (pH 8), 50 mM NaCl, 1% Triton X‐100, 0.1% sodium dodecyl sulfate (SDS), 0.5% sodium deoxycholate); phosphate‐buffered saline (PBS) with 2.5 mM MgCl2, 3 mM KCl and 0.5% or 1% Triton X‐100 (PBSMT0.5% or PBSMT1%), TAP lysis buffer (150 mM potassium acetate, pH 7.4, 20 mM HEPES‐KOH pH 7.4, 2 mM magnesium acetate, 1 mM EDTA, pH 8.0, 1 mM EGTA, pH 8.0, 0.5% or 1% Triton X‐100, 2 mM dithiothreitol, 1 mM PMSF and one mini EDTA‐free protease inhibitor cocktail tablet (Roche) per 50 ml; Blackwell and Brown, 2009) and UV‐crosslinking lysis buffer [ULB: 20 mM Tris pH 7.5, 50 mM LiCl, 1% SDS, 1 mM EDTA, 1% ß‐mercaptoethanol, 1 mg ml−1 heparin (Sigma‐Aldrich), 20 mM ribonucleoside vanadyl complexes (New England Biolabs)]. Cells were lysed with glass beads using a FastPrep FP120 (Thermo Savant) cell disruptor at 4°C (speed 6.5 for 30 s for all experiments except phosphatase treatment, which used 4 cycles of 4 s disruption), then mixed with additional lysis buffer. Crude lysate was obtained by centrifugation at 16,000g for 10 min at 4°C, and supernatants were clarified with an additional round of centrifugation under the same conditions.

Poly(A) RNA binding assay

To test whether Slr1 binds to polyadenylated mRNA, a UV crosslinking assay developed to detect mRNA‐binding proteins in S. cerevisiae (Marfatia et al., 2003) was adapted for n class="Species">C. albicans. Briefly, Slr1‐TAP‐expressing cells (SLR1‐TAP/slr1Δ; AMC94) were grown to mid‐log phase in 1 L YPD, washed and irradiated in a petri dish on ice with UV light in a Stratalinker 2400. Following lysis in ULB as described above, clarified lysates were adjusted to 0.5 M LiCl and bound to oligo (dT) cellulose resin (GE Healthcare) for 2 h at 4°C. After washing, oligo (dT)‐bound complexes were eluted and subjected to a second round of purification. To collect RNA–protein complexes, eluates were concentrated and precipitated prior to resuspension in RSB buffer (10 mM Tris, pH 7.4, 100 mM NaCl, 2.5 mM MgCl2, 1 mM CaCl2) with protease inhibitors. Following RNA digestion, the presence of Slr1‐TAP in oligo (dT)‐bound material was analyzed by immunoblotting with a horseradish peroxidase‐conjugated anti‐Protein A (PrA) antibody. Detailed methods are available in Supporting Information.

Identification of Slr1‐interacting proteins

To detect Slr1‐interacting proteins, SLR1‐TAP/slr1Δ (AMC94) and SLR1/slr1Δ (AMC91) cells were grown in 300 ml YPD + n class="Chemical">Uri to OD600 ∼ 2. Cells were lysed with glass beads in TAP lysis buffer as described above. Slr1‐TAP and interacting proteins were precipitated with immunoglobulin G (IgG)‐Sepharose (Pharmacia) at 4°C overnight, washed four times in TAP lysis buffer with 1% Triton X‐100 and 0, 0.5, 1 and 1.25 M potassium acetate, respectively, eluted with tobacco etch virus (TEV) protease (Invitrogen) (Blackwell and Brown, 2009), precipitated with trichloroacetic acid, and analyzed by SDS‐10% PAGE and blue silver (Candiano et al., 2004) or zinc staining (Bio‐Rad). Protein bands that were not precipitated from the untagged strain were excised from the blue‐silver stained gel for protein identification by mass spectrometry at The University of Texas Health Science Center at San Antonio.

To detect interactions with GFP‐tagged nuclear mRNA cap‐binding complex (CBC) proteins Cbc1 and Cbc2, orf19.387‐GFP/ORF19.387 SLR1‐TAP/slr1Δ (Cbc1‐GFP; AMC108) and orf19.763‐GFP/ORF19.763 SLR1‐TAP/slr1Δ (n class="Gene">Cbc2‐GFP; AMC109) cells were grown in 50 ml YPD to OD600 = 1–2 and lysed in TAP lysis buffer with 1% Triton X‐100 before IgG bead precipitation as described above. Following washing, Slr1‐TAP and interacting proteins were eluted with 3 M MgCl2, precipitated with trichloroacetic acid, and co‐precipitation of CBC proteins with Slr1‐TAP analyzed by anti‐GFP immunoblotting. For the RNase sensitivity experiment, lysates were pretreated with 10 µg ml−1 RNase A (Sigma‐Aldrich) for 15 min at 25°C. To test for background binding of Cbc1‐GFP and Cbc2‐GFP to the beads, orf19.387‐GFP/ORF19.387 SLR1/slr1Δ (Cbc1‐GFP; AMC106) and orf19.763‐GFP/ORF19.763 SLR1/slr1Δ (Cbc2‐GFP; AMC107) cells were employed for the pulldown experiment.

Phosphatase treatment

Cells expressing Slr1‐GFP (SLR1‐GFP/slr1Δ; AMC96) and slr1‐6SA‐GFP (slr1‐6SA‐GFP/slr1Δ; AMC99) as the sole copy of Slr1 were grown to mid‐log phase in medium lacking uridine at 30°C. Cells were lysed with glass beads in minimal RIPA buffer prior to resuspension in n class="Chemical">PBSMT0.5% buffer, as described above. Lysates (12 mg) were incubated with 4 µg anti‐GFP (Roche Diagnostics) for 1 h at 4°C. GFP fusion proteins were precipitated with 40 μl Protein G PLUS‐Agarose beads (Santa Cruz Biotechnology) by overnight incubation at 4°C. Beads were washed four times with PBSMT0.5% and divided into three aliquots, each resuspended in 50 μl NEB3 buffer. For each strain, one aliquot was incubated with 5 U calf intestinal phosphatase (New England Biolabs) at 37°C, and two controls were incubated without phosphatase at 0°C or 37°C. All samples were incubated for 60 min with agitation every 10 min. Slr1‐GFP proteins were eluted by boiling in SDS‐PAGE sample buffer. Purified proteins were resolved by SDS‐8% PAGE and analyzed by immunoblotting.

Identification of major arginine‐methylated proteins in C. albicans

Major arginine methylated proteins in n class="Species">Candida albicans were identified by immunoprecipitation with an anti‐dimethylarginine antibody. Cells from YPD cultures of hmt1Δ/Δ (AMC11), hmt1Δ/Δ + HMT1 (AMC14) and npl3Δ/Δ (AMC18) grown at 30°C to mid‐log phase were lysed in PBSMT1% as described above. Lysates (5 mg total protein) were incubated with 25 µl anti‐methylarginine antibody (Ab412; Abcam) overnight with rotation at 4°C. Protein G beads (40 µl packed volume; GE Lifesciences) were added to each lysate prior to 4 h incubation at 4°C with rotation. Beads were washed with PBSMT0.5% extensively prior to addition of protein sample buffer. Proteins isolated from 3 mg total protein were resolved by SDS‐12% PAGE and visualized by Coomassie staining. Major proteins not detected in cell lysates lacking the arginine methyltransferase were identified by mass spectrometry at the University of Texas San Antonio Health Sciences Center.

To confirm Slr1 methylation, mid‐log‐phase wildtype and n class="Gene">hmt1Δ/Δ cells expressing Slr1‐GFP (AMC85 and AMC86) were lysed in PBSMT0.5% as described above. Slr1‐GFP was precipitated from lysates (2.7 mg total protein) by incubation with 5 µl of monoclonal α‐GFP antibody (Roche Life Sciences) followed by protein G‐agarose incubation as above. The beads were washed in lysis buffer, and bound proteins eluted in SDS‐PAGE sample buffer, resolved by SDS‐10% PAGE and analyzed by immunoblotting.

Immunoblot analysis

GFP‐tagged proteins were recognized with a monoclonal α‐GFP antibody (Roche; 1:1000) and arginine methylation was recognized with a monoclonal α‐mono and dimethyl n class="Chemical">arginine antibody (Ab412, Abcam; 1:500), followed by a secondary α‐mouse‐horseradish peroxidase (HRP) antibody (GE Lifesciences; 1:5000). Slr1‐TAP was recognized with an HRP‐conjugated goat anti‐Protein A (PrA) antiserum (Rockland; 1:10,000). Ribosomal protein Rps3 was recognized with a polyclonal rabbit antibody against Rps3 (1:1000). Proteins were visualized through enhanced chemiluminescence (Pierce) and autoradiography.

Anti‐rabbit IgG (H + L)‐HRPO and anti‐n class="Species">mouse IgG (H + L) HRPO (Dianova) secondary antibodies were used for sucrose density gradient fraction immunoblots. The signals were detected with Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare) and the FUSION‐SL chemiluminescence detection system (Peqlab) and the Western blot analyses were quantified using the Image StudioLite software (LI‐COR).

Fluorescence microscopy

To visualize wildtype and mutant Slr1‐GFP proteins in n class="Species">yeast‐form cells, cells were grown to log phase in synthetic minimal medium lacking uridine (Uri−) at 30°C. Cells were washed with PBS and subsequently incubated with 10 µg ml−1 DAPI (4′,6‐diamidono‐2‐phenylindole; Molecular Probes) in PBS for 4 min at 30°C. Cells were washed with Uri− medium and visualized by Nomarski (differential interference contrast, DIC) and widefield epifluorescence microscopy (Olympus BX51: 100× objective, GFP and DAPI filters). Images were captured with an EvolutionVF color digital camera (noncooled, 12‐bit; MediaCybernetics) and QCapture Pro 5.0 software. Exposure times were the same for cells of all genotypes in the same experiment.

To visualize Slr1‐GFP in filamenting cells, cells were collected by centrifugation after 3 h growth in RPMI at 37°C, washed with n class="Chemical">PBS and visualized on agarose‐coated slides. To test for nuclear localization of wildtype and mutant Slr1‐GFP proteins in hyphal cells, cells were incubated for 1 min in 10 µg ml−1 DAPI in PBS prior to visualization. To determine whether slr1‐6SA‐GFP colocalized with the Spitzenkörper, after 3 h of hyphal induction in RPMI, FM4‐64 was added to slr1‐6SA‐GFP/slr1Δ (AMC99) and MLC1‐YFP/MLC1 (YJB7139) cells to a final concentration of 0.25 µg ml−1 and cultures incubated at 37°C for 4 min. Cells were rapidly collected by centrifugation, washed in PBS at 37°C and visualized immediately to allow detection of FM4‐64 in the Spitzenkörper. A Texas Red filter was used to detect FM4‐64 and a GFP filter was used to detect slr1‐6SA‐GFP and Mlc1‐YFP. Colocalization of the Spitzenkörper and GFP/YFP proteins was determined by stacking the FM4‐64 and GFP/YFP images in ImageJ v1.49 (Schneider et al., 2012), selecting the hyphal tip foci and noting overlap of selections between red and green images. Given the transitory nature of Spitzenkörper staining with FM4‐64, in one experiment 64–66 cells with distinct Spitzenkörper staining were analyzed and in additional experiments at least such 20 cells were analyzed. Line scans of FM4‐64, slr1‐6SA‐GFP and Mlc1‐YFP focal fluorescence that demonstrate partial colocalization of proteins with the Spitzenkörper at the hyphal tip are included in Supporting Information Fig. S4.

Fluorescence image analysis

To quantify subcellular localization of Slr1‐GFP proteins, the brightness/contrast levels of all DAPI images were first adjusted equivalently in Adobe Photoshop CS5 to lower background cytoplasmic fluorescence. GFP, n class="Chemical">DAPI and DIC images were then stacked in ImageJ v1.49 (Schneider et al., 2012) and cells with in‐focus nuclei were identified. The method of Hood‐DeGrenier (Hood‐DeGrenier et al., 2007) was modified to calculate two metrics of subcellular localization: percent nuclear fluorescence intensity (%N) and mean nuclear‐to‐cytoplasmic fluorescence intensity (N/C). For each cell, the whole cell and the nucleus were selected on DIC and DAPI images, respectively, and area and average pixel intensity (API) were measured for each selection on the GFP image. Selection was performed in triplicates for each cell using a Wacom Cintiq 13HD touch tablet; the mean of the three area and API measurements was used for subsequent calculations. For each image, the API of background regions lacking cells was subtracted from the whole‐cell and nuclear API measurements. The total nuclear and whole cell pixel intensities (TPIN and TPIWC) were calculated by multiplying the average area (A) by the background‐adjusted API. Percent nuclear fluorescence (%N) was calculated by dividing TPIN by TPIWC. The ratio of the API of the nucleus to the API of the cytoplasm (N/C) was calculated as follows: N/C = APIN/[(TPIWC − TPIN)/(AWC − AN)]. Three independent experiments were performed for each set of strains and the data analyzed in R (R Core Team, 2016). Differences among cells of different genotypes in a single experiment were evaluated by the Kruskal‐Wallis rank sum test (p < 0.0001 for all experiments), given the non‐normal distribution of the data. Pairwise Mann–Whitney–Wilcoxon tests with Bonferroni correction for multiple comparisons were then used to determine whether the localization of specific Slr1‐GFP proteins differed from each other. Box plots were generated with GraphPad Prism 7.0 software. The presence of slr1‐6SA‐GFP foci at the bud neck of post‐mitotic cells was quantified in three trials (> 55 post‐mitotic cells per genotype).

To quantify localization of slr1‐6SA to the hyphal tip in cells with and without n class="Gene">She3, two independent cultures each of she3Δ/SHE3 slr1‐6SA‐GFP/SLR1 (AMC138) and she3Δ/she3Δ slr1‐6SA‐GFP/SLR1 (AMC139) were induced to filament in RPMI and prepared for microscopy as above. GFP and DIC images were overlaid and hyphal cells were divided into two categories: cells with the most distal slr1‐6SA‐GFP focus (a) within 2 µm (one hyphal width) of the hyphal tip or (b) farther than 2 µm from the tip. Exposure times were equivalent for all strains in a single experiment and images were overlaid using Adobe Photoshop CS5. At least 65 hyphal cells of each genotype were analyzed in three independent trials. Differences in hyphal tip localization of slr1‐6SA‐GFP between cells with and without She3 were evaluated by an unpaired student's t‐test.

Fluorescence in situ mRNA hybridization

A protocol to detect localization of bulk mRNA in S. cerevisiae was adapted for use in n class="Species">C. albicans cells (Green et al., 2002). Wildtype (AMC79) and slr1Δ/Δ (AMC89) cells were collected from 1 mL synthetic dropout culture (OD600 ∼ 0.2) grown at 30 or 37°C. For heat shock, cells were grown at 30°C to ∼ 0.2 OD600 and shifted to 42°C for 30 min. Cells were then fixed in 5% formaldehyde for 1 h, washed twice in P solution (1.2 M sorbitol in 0.1 M potassium phosphate buffer, pH 6.8), and resuspended in 1 ml P solution. Cells from 20 μl suspension were placed on a multiwell slide coated with 0.1% poly‐L‐lysine (Sigma‐Aldrich), spheroplasted in P solution containing 500 μg ml−1 Zymolyase (100T, US Biological) and 1% β‐mercaptoethanol, permeabilized by addition of 0.5% Triton X‐100 in P solution for 10 min, equilibrated with 0.1 M triethanolamine (pH 8.0), and polar groups blocked with 0.25% acetic anhydride. Cells were incubated in prehybridization buffer (1 mg ml−1 yeast tRNA (Sigma‐Aldrich), 0.005% BSA, 10% dextran sulfate, 25% deionized formamide and 2× SSC) and hybridized overnight with 250 nM digoxigenin‐labeled oligo(dT) probe (IDT). Cells were washed with 2× and 4× SSC, incubated with FITC‐conjugated anti‐digoxigenin antibody (Roche; 1:200 dilution in 2× SSC) at room temperature for 1–2 h, and briefly stained with DAPI and washed with 2× SSC. Cells were examined by Nomarski (DIC) and fluorescence microscopy as described above.

Sucrose‐density fractionation experiments

The preparation and fractionation of sucrose‐density gradients was carried out in three replicate experiments following published protocols for n class="Species">S. cerevisiae, modified for use with C. albicans (Gross et al., 2007; Baierlein et al., 2013). Briefly, 300 ml SLR1‐GFP/slr1Δ (AMC96) yeast cell cultures were grown to log phase in YPD at 30°C. Cycloheximide (Sigma‐Aldrich) was added to a final concentration of 100 µg ml−1 and cells were incubated for 15 min on ice. After harvesting, the cell pellets were lysed with the same amount of glass beads in lysis buffer (20 mM HEPES‐KOH pH7.5, 10 mM KCl, 2.5 mM MgCl2, 1 mM EGTA, 1 mM DTT, 100 µg ml−1 cycloheximide) supplemented with Complete, EDTA‐free protease inhibitor cocktail (Roche). Cells were lysed with 5 cycles of 1.5 min in a BeadBeater (Biospec) followed by 2 min on ice. The lysates were centrifuged at 4°C once for 5 min at 16,000g and the supernatant cleared by additional centrifugation for 10 min at 16,000g. If indicated, the lysates were treated with 0.25 mg ml−1 RNase A (AppliChem) for 20 min on ice. For protein and ribosomal profile analyses 30 OD260 nm units of lysates were loaded onto a linear 7%–47% (w/v) sucrose gradients (20 mM HEPES‐KOH pH 7.5, 10 mM KCl, 2.5 mM MgCl2, 1 mM EGTA) poured with the Gradient Master machine (Biocomp) and centrifuged for 3 h at 40,000 rpm and 4°C in a TH‐641 rotor and Sorvall WX80 ultracentrifuge (Thermo Scientific). The gradients were fractionated with a density‐gradient fractionator (Teledyne Isco) while the absorbance at 254 nm was documented.

Protein fractions were precipitated with 10% trichloroacetic acid (n class="Chemical">TCA), washed two times with 70% acetone and subjected to SDS‐PAGE and Western blotting. To be able to load the whole gradient on one gel and to increase the signal strength, some fractions were pooled as indicated.

Author contributions

C.A. and A.M. made major contributions to the conception and design of this study. C.A., A.M., C.B., X.L., S.L., O.M., P.M., K.O., T.P., S.S. and H.K. contributed to the acquisition, analysis and interpretation of the data. A.M., C.A., C.B. and X.L. contributed to the writing of the manuscript.

Supporting Information

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