A C-terminal domain conserved in precursor processing proteases is required for intramolecular N-terminal maturation of pro-Kex2 protease.

Abstract

The Kex2 protease of the yeast Saccharomyces cerevisiae is the prototype of a family of eukaryotic subtilisin homologs thought to process prohormones and other precursors in the secretory pathway. Deletion analysis of Kex2 protease shows that a sequence of 154‐159 residues carboxyl to the subtilisin domain is essential for the formation of active enzyme. Disruption of this region, termed the ‘P‐domain’, blocks the normally rapid intra‐molecular cleavage of the N‐terminal pro‐segment of pro‐Kex2 protease in the endoplasmic reticulum (ER). The C‐terminal boundary of the P‐domain coincides closely with the endpoint of similarity between Kex2 protease and its mammalian homologues. The conservation of and functional requirement for the P‐domain sharpens the distinction between a ‘Kex2 family’ of processing enzymes and degradative ‘subtilases’, and implies that the Kex2‐related enzymes have in common entirely novel structural features that are important in the maturation of precursor polypeptide substrates. Failure to cleave the N‐terminal pro‐domain, due either to truncation of the P‐domain or to mutation of the active site histidine or serine, results in stable, intracellular retention of pro‐enzyme, apparently in the ER. Thus pro‐Kex2 protease appears to contain an ER retention signal which is removed or destroyed by cleavage of the pro‐domain.

Mutational analysis of predicted interactions between the catalytic and P domains of prohormone convertase 3 (PC3/PC1)

“…These data reveal that PC3D616-EGFP can be sorted normally into the regulated secretory pathway, whereas PC3⌬P-EGFP cannot, but instead accumulates in the ER. This finding is thus consistent with earlier observations that PC3 cannot fold without a P domain (33,36,40). Full-length or WT PC3 consists of a signal peptide (Pre) and four domains, pro-region (Pro), catalytic domain (Cat), P domain (P), and C-terminal domain.…”

“…We propose that this charge imbalance may lead to instability in the catalytic domain, which is offset by interaction of the P domain with the catalytic domain to provide stabilization. Indeed, several truncation experiments have shown the P domains are required for the stable folding of the SPCs (33,36,40). In those prior studies, P domain-deleted convertases were expressed in cells, but were unable to traverse the secretory pathway, an indication of misfolding.…”

Functional analysis of human PACE4‐A and PACE4‐C isoforms: identification of a new PACE4‐CS isoform

“…From the reported importance of the pro-segment acting as an intramolecular chaperone in subtilisin-like enzymes [14], it was suggested that PACE4-D would code for an inactive enzyme [10]. Similarly, as the integrity of the P-domain has been reported to be critical for the activity of yeast kexin [15] and mouse furin [16], it was also predicted that PACE4-B, which lacks the P-domain, would be inactive [9]. In contrast, PACE4-C (652 aa), which is truncated at the C-terminus compared to PACE4-A (969 aa), has a distinct sequence at the end of the P-domain, resulting in a shorter form in which the 32 amino acids following Gly 620 are different.…”

“…In contrast, PACE4-C (652 aa), which is truncated at the C-terminus compared to PACE4-A (969 aa), has a distinct sequence at the end of the P-domain, resulting in a shorter form in which the 32 amino acids following Gly 620 are different. While the C-terminal border of the P-domain has been suggested to be close to Gly 631 [5,15,16], its exact site is not known with certainty and could be convertase-dependent. Therefore, it was suggested that PACE4-C could code for an active convertase [10].…”

Binding of BiP to the Processing Enzyme Lymphoma Proprotein Convertase Prevents Aggregation, but Slows Down Maturation

“…This process has been found to be autocatalytic and intramolecular for furin (15,16), PC1 (17)(18)(19), PACE4 (20), and LPC (10). Propeptide cleavage is a prerequisite for exit out of the endoplasmic reticulum (ER) and requires the presence of both the middle and catalytic domains (21)(22)(23)(24). The requirement for sequences in the middle domain was illustrated recently in a patient with a multihormonal syndrome including severe childhood obesity.…”