Processing of predicted substrates of fungal Kex2 proteinases from Candida albicans, C. glabrata, Saccharomyces cerevisiae and Pichia pastoris.
BACKGROUND
Kexin-like proteinases are a subfamily of the subtilisin-like serine proteinases with multiple regulatory functions in eukaryotes. In the yeast Saccharomyces cerevisiae the Kex2 protein is biochemically well investigated, however, with the exception of a few well known proteins such as the alpha-pheromone precursors, killer toxin precursors and aspartic proteinase propeptides, very few substrates are known. Fungal kex2 deletion mutants display pleiotropic phenotypes that are thought to result from the failure to proteolytically activate such substrates.


RESULTS
In this study we have aimed at providing an improved assembly of Kex2 target proteins to explain the phenotypes observed in fungal kex2 deletion mutants by in vitro digestion of recombinant substrates from Candida albicans and C. glabrata. We identified CaEce1, CA0365, one member of the Pry protein family and CaOps4-homolog proteins as novel Kex2 substrates.


CONCLUSIONS
Statistical analysis of the cleavage sites revealed extended subsite recognition of negatively charged residues in the P1', P2' and P4' positions, which is also reflected in construction of the respective binding pockets in the ScKex2 enzyme. Additionally, we provide evidence for the existence of structural constrains in potential substrates prohibiting proteolysis. Furthermore, by using purified Kex2 proteinases from S. cerevisiae, P. pastoris, C. albicans and C. glabrata, we show that while the substrate specificity is generally conserved between organisms, the proteinases are still distinct from each other and are likely to have additional unique substrate recognition.
Bader
Oliver
O
FG16, Robert Koch-Institut, Nordufer 20, D-13353 Berlin, Germany. obader@gwdg.de
Krauke
Yannick
Y
Hube
Bernhard
B
D000595:Amino Acid Sequence; D002176:Candida albicans / Q000201:enzymology*; D041221:Candida glabrata / Q000201:enzymology*; D015966:Gene Expression Regulation, Fungal; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010641:Phenotype; D010843:Pichia / Q000201:enzymology*; D043484:Proprotein Convertases / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D013379:Substrate Specificity
D029701:Saccharomyces cerevisiae Proteins; D043484:Proprotein Convertases; C058751:KEX2 protein, S cerevisiae