Solubilization and characterization of yeast signal peptidase.
An efficient post-translational assay for solubilized yeast signal peptidase has been developed. The enzyme can be solubilized in nonionic detergent (0.5% Nikkol) without added salt, but salt increased the efficiency of solubilization. Radiosequencing of the cleaved substrate revealed that the enzyme removed the signal peptide. The substrate (prepro-alpha-factor) must be pretreated with sodium dodecyl sulfate to be cleaved. The enzyme displays a broad, alkaline pH optimum, retaining activity at pH 12. Moderately high temperatures (35 degrees C), excess detergent (greater than 0.5% Nikkol), or high salt (greater than 300 mM KOAc) will inactivate the enzyme. Phosphatidylcholine is necessary for optimal activity. The optimal ratio of Nikkol:lipid:sodium dodecyl sulfate is 6.4:2.2:1. The membrane association of yeast signal peptidase is resistant to carbonate extraction, indicating that it is an integral membrane protein.
YaDeau
J T
JT
Laboratory of Cell Biology, Howard Hughes Medical Institute, Rockefeller University, New York, New York 10021.
Blobel
G
G
D010450:Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics; D007700:Kinetics; D008565:Membrane Proteins; D008861:Microsomes / Q000201:enzymology; D010944:Plants / Q000235:genetics; D010957:Plasmids; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D012697:Serine Endopeptidases; D012995:Solubility; D014908:Triticum / Q000235:genetics
D005656:Fungal Proteins; C048632:MF(ALPHA)1 protein, S cerevisiae; D008565:Membrane Proteins; D011498:Protein Precursors; D029701:Saccharomyces cerevisiae Proteins; D010450:Endopeptidases; D012697:Serine Endopeptidases; C026515:type I signal peptidase