EXTENSIVE SEQUENCING OF TRYPTIC PEPTIDES OF A RABBIT RETICULOCYTE 66-KDA PROTEIN THAT PROMOTES RECYCLING OF HSP-70 - HOMOLOGY TO STRESS-RELATED PROTEINS

Trypsinization and sequence analysis of the 66-kDa rabbit reticulocyte protein (RF-hsp 70), shown in the preceding article to function as a recycling protein for hsp 70, demonstrates striking similarity to the transformation-sensitive human protein IEF SSP 3521 (Honore, B., Leffe rs, H., Madsen, P., Rasmussen, H. H., Vandekerckhove, J., and Cells, J . E. (1992) J. Biol. Chem. 267, 8485-8491) and mouse extendin (Blatch, G.L., Lassie, M., Takatori, T., Gandhi, T., Kundra, V., and Zetter, B . R. (1995) Proc. Am. Assoc. Cancer Res. 36, 68). The human and mouse proteins share 97% sequence identity, and sequencing of 20 polypeptide s (225 residues) from RF-hsp 70 reveals only 10 differences be tween t he rabbit and human proteins and 13 differences between the rabbit and mouse proteins (96 and 94% identity, respectively). In addition, all three proteins are of similar size, and each contains 11 cysteines. Th ese findings strongly suggest that these three proteins are homologs o f the same activity. All differences (but one) between the human and m ouse proteins occur within the amino-terminal half of the protein, and there is only one difference among 121 sequenced residues between RF- hsp 70 and the human or mouse protein which occurs within the carboxyl -terminal 70% of the molecule. In addition, where partial sequences of RF-hsp 70 and p60, a chick oviduct protein that shows 70% identity to the human protein (Smith, D. F., Sullivan, W. P., Marion, T. N., Zait su, K., Madden, B., McCormick, D. J., and Toft, D. O., (1993) Mol. Cel l. Biol. 13, 869-876), overlap (a total of 54 residues), RF hsp 70 and chick p60 show 78% sequence identity, Studies of the initial digestio n of RF-hsp 70 by trypsin indicate that it is first converted to 58- a nd 54-kDa components, each of which is then converted to a 43-kDa poly peptide. This 43-kDa component is located in the human and mouse prote ins at position 124 to about 470. It is converted subsequently to a 31 -kDa polypeptide by trypsin hydrolysis at position 207. This 31-kDa co mponent is finally split into 17- and 14-kDa polypeptides that are loc ated at positions 208 to approximately 351 and 352 to approximately 47 0, respectively. The 14-kDa polypeptide is relatively resistant to fur ther digestion with trypsin, and seven tryptic peptides from other par ts of RF-hsp 70 contain internal lysine and/or arginine residues (as d o several tryptic peptides produced from IEF SSP 3521 and chick p60). Both features may be due to interference with trypsin action by second ary structure in the protein, since trypsinization of reduced and carb oxymethylated RF-hsp 70 results in hydrolysis of the 14-kDa polypeptid e and reduces the level of peptides that contain internal lysine and/o r arginine, although it does not eliminate them.