trans-Complementation assay establishes the role of proregion hydrophobic amino acid residues in the biosynthesis of Saccharomyces cerevisiae Kex2p endoprotease.
The proregion of Saccharomyces cerevisiae endoprotease Kex2p is essential for the biosynthesis of an active enzyme. It has been suggested that the proregion acts in the endoplasmic reticulum to catalyse folding of the enzyme. To identify amino acid residues important for proregion function, we used an in vivo system in which the Kex2p proregion can act in trans to activate a Kex2p enzyme synthesized without its proregion. Activation of Kex2p by wild-type and mutated proregions revealed the essential role of hydrophobic residues F(37), V(39) and F(70) in enzyme activation. Further exploration of the role of these residues by in vitro inhibition of Kex2p activity by its proregion indicated that they are essential to form the proregion/enzyme bimolecular complex. In contrast, basic residues K(108) and R(109), located in the C-terminus of the proregion, are not involved in complex formation but are necessary for the biosynthesis of an active enzyme.
Lesage
Guillaume
G
Département de Biochimie, Université de Montréal, Montréal (Québec) H3C 3J7, Canada.
Guimond
Julie
J
Boileau
Guy
G
D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000235:genetics / Q000378:metabolism*; D005816:Genetic Complementation Test; D057927:Hydrophobic and Hydrophilic Interactions; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D043484:Proprotein Convertases; D017510:Protein Folding; D011499:Protein Processing, Post-Translational / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013381:Subtilisins / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D013499:Surface Properties
D004792:Enzyme Precursors; D029701:Saccharomyces cerevisiae Proteins; D043484:Proprotein Convertases; D013381:Subtilisins; C058751:KEX2 protein, S cerevisiae