The core proteins of 35S hnRNP complexes. Characterization of nine different species.

Ribonucleoprotein complexes (hnRNP) containing fragments of heterogeneous nuclear (hn)RNA and sedimenting at 35-40 S were isolated from the nuclei of HeLa S3 cells using the pH 8.0/diffusion technique. These hnRNP complexes are thought to be part of the hnRNA processing apparatus. The major protein components (core proteins) were identified by their constant ratios in native particles and in 35S hnRNP particles reconstituted in vitro. All of the core proteins, with one exception, show an increase in Mr on sodium dodecylsulfate (NaDodSO4)/polyacrylamide gels containing 8 M urea, indicative of secondary structure elements resistant to denaturation by NaDodSO4. The nine core proteins found by us are: A1 [Mr(NaDodSO4) 31 X 10(3)/Mr (urea) 38 X 10(3), apparent isoelectric point, pIapp 9.3], A2 (32.5 X 10(3)/39 X 10(3), 8.4), B1a (35.5 X 10(3)/41 X 10(3), 8.8), B1b (35.5 X 10(3)/44 X 10(3), 8.3), B1c (35.5 X 10(3)/43 X 10(3), 5.7) B2 (37 X 10(3)/42 X 10(3), 9.15), C1 (39 X 10(3)/46 X 10(3), 9.2), C2 (40.5 X 10(3)/45 X 10(3), 5.55) and C3 (38.5 X 10(3)/37 X 10(3), 4.8). Individual proteins were electroeluted from two-dimensional gels and their amino acid composition determined. Difference indices were calculated and show a group of closely related basic proteins (A1, A2, B1a, B1b, B2, C1), two related slightly acidic proteins (B1c, C2) and a distinct acidic member (C3). Two-dimensional analysis of tryptic fragments and one-dimensional separation of peptides after V8 protease treatment support these data. Peptide mapping of the proteins A1 and A2 from bovine and human cells yields identical fragments indicating a high degree of cross-species conservation. An additional protein (D4: 44 X 10(3)/55 X 10(3), greater than 9.5) was found, which preferentially associates with heavier, oligomeric hnRNP structures. Only traces of actin are present in the 35S hnRNP fraction. All core proteins are modified by charge. A large part of the charge isomers arises by phosphorylation, which has been shown by labeling with 32PO4 in vivo and with [gamma-32P]ATP in vitro. In vitro the phosphate transfer is mediated by an endogenous protein kinase associated with the 35S hnRNP complexes. The major core protein A1 exists in two conformeric forms (A1 and A1x) of which only A1x serves as phosphate acceptor in vivo.