Dileucine signal-dependent and AP-1-independent targeting of a lysosomal glycoprotein in Trypanosoma brucei.

Sorting of trans-membrane proteins destined for the lysosome is achieved by selective inclusion into post-Golgi transport vesicles. In higher eukaryotes sorting may be mediated by a peptidic motif, principally acidic clusters and tyrosine- or dileucine-based cytoplasmic signals or by inclusion of mannose-6-phosphate (M6P) into the N-glycans of lysosomal proteins. In African trypanosomes a major lysosomal trans-membrane protein is CB-1/p67. The cytoplasmic domain of p67 lacks tyrosine and lysine, but does contain a canonical dileucine sequence embedded within an acidic region. AP-1, -3 and -4 adaptin complexes, which recognise tyrosine- and dileucine-sorting signals, are encoded by the trypanosome genome, but the genes for M6P-receptors or activities required to produce M6P are absent, suggesting that lysosomal delivery of p67 is most likely adaptin-mediated. By construction of p67 reporter constructs we show that the dileucine signal is necessary and sufficient for efficient lysosomal delivery of a trans-membrane protein in bloodstream stage trypanosomes. However, this targeting does not require AP-1, as knockdown of the trypanosome gamma-adaptin subunit by RNAi has no detectable effect on the location or maturation of p67. These data suggest that p67 is targeted to the lysosome by dileucine-dependent but AP-1-independent mechanisms.