Posttranslational processing of the prohormone-cleaving Kex2 protease in the Saccharomyces cerevisiae secretory pathway.

Wilcox, Celeste A.; Fuller, Robert S.

doi:10.1083/jcb.115.2.297

Cited by 116 publications

(100 citation statements)

References 45 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Likewise, membrane proteases, such as signal peptidase and the KEX2p of yeast, have been implicated in the processing of secretory and membrane proteins (33,35,36). In this work we show that ER membranes, free of soluble lumenal contents, retain the proteases necessary degrade HMG-CoA reductase in a mevalonate-or sterol-stimulated, ALLN-sensitive fashion (Fig.…”

Section: Figmentioning

confidence: 58%

Degradation of 3-Hydroxy-3-methylglutaryl-CoA Reductase in Endoplasmic Reticulum Membranes Is Accelerated as a Result of Increased Susceptibility to Proteolysis

McGee

Cheng

Kumagai

et al. 1996

Journal of Biological Chemistry

View full text Add to dashboard Cite

The endoplasmic reticulum (ER) membrane protein 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase is subject to regulated degradation when cells are presented with an excess of sterols or mevalonate. In this report, we demonstrate the degradation of HMGCoA reductase in ER membranes prepared from cells which have been pretreated with mevalonate or sterols prior to membrane purification. Degradation of HMGCoA reductase in membranes prepared from pretreated cells is more rapid than in membranes prepared from cells which have received no regulatory molecules. In vitro degradation is blocked by protease inhibitors previously shown to inhibit reductase degradation in vivo and is specific for intact HMG-CoA reductase. The lumenal contents of the ER membranes are dispensible for the regulated proteolysis and the proteases responsible for reductase degradation are stably associated with the ER membrane. Regulated proteolysis of HMG-CoA reductase is inhibited by lactacystin, a newly defined inhibitor of the multicatalytic protease, the proteasome, and in vitro degradation of reductase correlates with the presence of proteasome subunits in purified ER membranes. The ubiquitin system for protein degradation, which has recently been shown to be required for the degradation of several ER membrane proteins, is not required for the degradation of HMG-CoA reductase. Finally, we conclude that the regulated proteolysis of HMG-CoA reductase in response to regulatory molecules such as mevalonate or sterols is mediated by increased susceptibility of the reductase to ER proteases, rather than the induction of a new proteolytic activity.

show abstract

Section: Figmentioning

confidence: 58%

Degradation of 3-Hydroxy-3-methylglutaryl-CoA Reductase in Endoplasmic Reticulum Membranes Is Accelerated as a Result of Increased Susceptibility to Proteolysis

McGee

Cheng

Kumagai

et al. 1996

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Previous work established that the proregions of a number of convertases in the subtilisin-like family must be cleaved to activate the endoprotease and to allow transport out of the endoplasmic reticulum [21,30,44,[52][53][54][55][56]. However, when expressed in AtT-20 or hLoVo cells, PC1-pro-PACE4 processing was indistinguishable from that of native PACE4, and the secretion of the resultant mature PACE4 was not improved (not shown).…”

Section: Role Of the N-terminal Proregion In Pace4 Biosynthesismentioning

confidence: 98%

PACE4: a subtilisin-like endoprotease with unique properties

Mains

Berard

Denault

et al. 1997

Biochemical Journal

View full text Add to dashboard Cite

PACE4 is one of the neuroendocrine-specific mammalian subtilisin-related endoproteases believed to function in the secretory pathway. The biosynthesis and secretion of PACE4 have been studied using transfected neuroendocrine and fibroblast cell lines, as well as primary pituitary cultures. ProPACE4 (approx. 106 kDa) is cleaved intracellularly before secretion of PACE4 (approx. 97 kDa); the N-terminal propeptide cleavage is accelerated in a truncated form of PACE4 lacking the Cys-rich C-terminal region (PACE4s). Neither PACE4 nor PACE4s is stored in regulated neuroendocrine secretory granules, whereas pro-opiomelanocortin-derived peptides and prohormone convertase 1 enter the regulated secretory pathway efficiently. The relatively slow cleavage of the proregion of proPACE4 in primary anterior pituitary cells, followed by rapid secretion of PACE4, is similar to the results for proPACE4 in transfected cell lines. The enzyme activity of PACE4 is distinct from furin and prohormone convertases, both in the marked sensitivity of PACE4 to inhibition by leupeptin and the relative insensitivity of PACE4 to inhibition by Ca2+ chelators and dithiothreitol; PACE4 is not inhibited by the α1-antitrypsin Portland variant that is very potent at inhibiting furin. The unique biosynthetic and enzymic patterns seen for PACE4 suggest a role for this neuroendocrine-specific subtilisin-like endoprotease outside the pathway for peptide biosynthesis.

show abstract

“…In vivo, the enzyme is concentrated 30-fold relative to other secretory proteins (16) in a late Golgi compartment (33,34) where it might function as part of an assembly of processing enzymes. Native substrates may be cleaved with different kinetics than corresponding synthetic substrates due to substrate secondary structure or interactions distant from or C-terminal to the cleavage site.…”

Section: Discussionmentioning

confidence: 99%

Structural and enzymatic characterization of a purified prohormone-processing enzyme: secreted, soluble Kex2 protease.

Brenner

Fuller

1992

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

177

145

View full text Add to dashboard Cite

The prohormone-processing Kex2 protease of the budding yeast Saccharomyces cerevisiae can be converted from an intracellular membrane protein to a soluble, secreted, and active form by deletion of the transmembrane domain and C-terminal tail. One such molecule was purified to near homogeneity from the culture medium of an overexpressing yeast strain. Amino acid sequence analysis revealed that the N terminus of mature Kex2 protease is created by a potentially autoproteolytic cleavage at Lys'"-Arg, prior to the domain homologous to subtilisin, followed by trimming of Leu-Pro and Val-Pro dipeptides by the Stel3 dipeptidyl aminopeptidase. Kinetic parameters were examined using fluorogenic peptidylmethylcoumarin amide substrates. Initial burst titration indicated that the preparation was entirely active. Measurements of dependence of activity on pH yielded a simple curve suggesting titration of a single ionizable group. Activity was half-maimal at pH 5.7 and nearly constant from pH 6.5 to 9.5. Discrimination between substrates was as great as 360-fold in Km and 130-fold in kt. Substrates with a Lys-Arg dipeptide preceding the cleaved bond were preferred, having ktlKm values up to 1.1 x 107 sec-1 M-'. The enzyme cleaved substrates having Arg-Arg, Pro-Arg, Ala-Arg, and Thr-Arg with increased Km but with unchanged k,,t. In contrast, the enzyme displayed a dramatically lower kt for a Lys-Lys substrate with a smaller increase in K. Thus the two residues preceding the cleaved bond may play distinct roles in the selectivity ofbinding and cleavage of probormone substrates.The Saccharomyces cerevisiae Kex2 protein, a Ca2+-dependent serine protease, is the only enzyme proven by genetic and biochemical evidence to cleave a prohormone (pro-a-factor) at pairs of basic residues in the eukaryotic secretory pathway (1-3). The high specificity of Kex2 protease, homologous to the subtilisin family (4, 5), contrasts with the broad specificity of previously known members of the family, all of which are degradative enzymes. Kex2 and its mammalian homologues furin (5, 6), PC2 (7,8) MATERIALS AND METHODS Expression System. ss-Kex2 was purified from the culture media of strains CB023 and KRY77-3B carrying plasmid pG5-KEX2AC3 (Fig. 1)

show abstract

Posttranslational processing of the prohormone-cleaving Kex2 protease in the Saccharomyces cerevisiae secretory pathway.

Cited by 116 publications

References 45 publications

Degradation of 3-Hydroxy-3-methylglutaryl-CoA Reductase in Endoplasmic Reticulum Membranes Is Accelerated as a Result of Increased Susceptibility to Proteolysis

Degradation of 3-Hydroxy-3-methylglutaryl-CoA Reductase in Endoplasmic Reticulum Membranes Is Accelerated as a Result of Increased Susceptibility to Proteolysis

PACE4: a subtilisin-like endoprotease with unique properties

Structural and enzymatic characterization of a purified prohormone-processing enzyme: secreted, soluble Kex2 protease.

Contact Info

Product

Resources

About