Abstract
Canine microsomal signal peptidase activity has been shown previously to co-migrate as an apparent complex of six polypeptides with molecular masses of 25, 23, 22, 21, 18, and 12 kDa. The 22- and 23-kDa species are differentially glycosylated forms of the same protein, designated SPC 22/23. The amino acid sequence of SPC 22/23 was deduced from cDNA clones. The protein is synthesized without a cleavable amino-terminal signal sequence and contains a single site for N-linked glycosylation. SPC 22/23 appears to be anchored to the rough endoplasmic reticulum membrane by a single hydrophobic segment near its amino terminus, with the remainder of the protein positioned on the lumenal side of the membrane. The amino acid sequence of SPC 22/23 shares homology with tryptic peptides derived from the hen oviduct signal peptidase glycoprotein, one of two possible proteins required for signal peptide processing in the avian system (Baker, R.K., and Lively, M.O. (1987) Biochemistry 26, 8561-8567). Therefore, the complete amino acid sequence of SPC 22/23 presented in this report corresponds to one of two possible proteins required for signal peptide processing in higher eukaryotic cells.
Highlights
The physical association between signal peptidase complex (SPC) proteins was suggested because of their co-migration through multiple rounds of chromatography and co-sedimentation during sucrose gradient centrifugation (Evans et al, 1986a).It was subsequently found that the 22- and 23-kDa signal peptidase complex proteins possess identical amino-terminal amino acid sequences (Evans et al, 198613)
In an attempt to define the function of SPC 22/23, the glycoprotein subunit of the canine signal peptidase complex, its amino acid sequence was deduced from DNA clones and compared with other previously sequenced proteins, including those involved in signal peptide processing in bacteria and yeast
No sequence similarity exists between SPC 22/23 and theyeast SECl I protein or the E. coli processing enzymes, it is interesting to note otherstructural similarities
Summary
0 1988 by The American Society for Biochemistry and Molecular Biology, Inc. Vol 263, No 32, Issue of November 15, pp. 17063-17070,1988 Printed in U.S.A. The physical association between signal peptidase complex (SPC) proteins was suggested because of their co-migration through multiple rounds of chromatography and co-sedimentation during sucrose gradient centrifugation (Evans et al, 1986a).It was subsequently found that the 22- and 23-kDa signal peptidase complex proteins possess identical amino-terminal amino acid sequences (Evans et al, 198613) This result, coupled with the finding that both proteins are reduced to a single 19-kDa electrophoretic band upon treatment with endoglycosidase H (Evans et al, 1986a), indicated that the 22- and 23-kDa polypeptides are differentially glycosylated forms of the same protein, designated SPC 22/23. Cod-SepharoseAffinity Chromatography of Solubilized Membranes-It was previously reported (Evans et al, 198613;Evans, 1986) that the entire signal peptidase complex binds to ConA-Sepharose, only the 22- and 23-kDa constituent proteins are N-glycosylated This property of the SPC was exploited for purification of the signal peptidase complex from detergent-solubilizedextracts. The column was eluted with 20 ml of 0.6 M sodium phosphate, pH 6.8,0.1% SDS, 1mM EDTA, 1mM DTT
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