Abstract
Canine microsomal signal peptidase activity was previously isolated as a complex of five subunits (25, 22/23, 21, 18, and 12 kDa). Two of the signal peptidase complex (SPC) subunits (23/23 and 21 kDa) have been cloned and sequenced. One of these, the 21-kDa subunit, was observed to be a mammalian homolog of SEC11 protein (Sec11p) (Greenburg, G., Shelness, G. S., and Blobel, G. (1989) J. Biol. Chem. 264, 15762-15765) a gene product essential for signal peptide processing and cell growth in yeast (Böhni, P.C., Deshqies, R.J., and Schekman, R.W. (1988) J. Cell Biol. 106, 1035-1042). cDNA clones for the 18-kDa SPC subunit have now been characterized and found to encode a second SEC11p homolog. Both the 18- and 21-kDa canine SPC subunits are integral membrane proteins by virtue of their resistance to alkaline extraction. Upon detergent solubilization, both proteins are found in a complex with the 22/23 kDa SPC subunit, the only SPC subunit containing N-linked oligosaccharide. No steady-state pool of canine Sec11p-like monomers is detected in microsomal membranes. Alkaline extraction of microsomes prior to solubilization or solubilization at alkaline pH causes partial dissociation of the SPC. The Sec11p-like subunits displaced from the complex under these conditions demonstrate no signal peptide processing activity by themselves. The existence of homologous subunits is common to a number of known protein complexes and provides further evidence that the association between SPC proteins observed in vitro may be physiologically relevant to the mechanism of signal peptide processing and perhaps protein translocation.
Highlights
CDNA clones for the 1%kDa SPC subunit have been characterized and found to encode a second SECllp homolog
21-kDa canine SPC subunits are integral membrane proteins by virtue of their resistance to alkaline extraction. Both proteins are found in a complex with the 22/23kDa SPC subunit, the only SPC subunit containing N-linked oligosaccharide
The Secllp-like subunits displaced from the complex under these conditions demonstrate no signal peptide processing activity by themselves
Summary
Materials-Reagents were obtained from the following sources: avian myeloblastosis virus reverse transcriptase, Life Sciences, Inc.; restriction enzymes, Boehringer Mannheim or New England Bio-. Five ml of washed microsomes (2 eq/pl) in RM buffer were diluted lo-fold to a final concentration of 0.1 M sodium carbonate, pH 11.5, and incubated on ice for 15 min. Solubilization of sodium carbonate-extracted microsomes (Fig. 8, condition 1) was performed as described, except that homogenization was performed at room temperature, followed by a lo-min incubation at 15 “C (Baker et al, 1986). Solubilization of salt- and EDTA-washed rough microsomes (5 ml, 2 eq/pl) at pH 11 (Fig. 8, condition 2) was performed as described previously (Shelness et al, 1988), except that the final solubilization mixture contained 0.5 M sodium carbonate, pH 11.5, instead of 0.5 M KOAc, pH 7.5, and no triethanolamine buffer. The void volume fractions were pooled, aliquoted, and stored at -20 “C. The reaction conditions used to convert preprolactin to prolactin were identical to those of Evans et al (1986a), except that the final DTT concentration was 20 mM (Baker and Lively, 1987)
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