Ste24p Mediates Proteolysis of Both Isoprenylated and Non-prenylated Oligopeptides

Abstract

Rce1p and Ste24p are integral membrane proteins involved in the proteolytic maturation of isoprenylated proteins. Extensive published evidence indicates that Rce1p requires the isoprenyl moiety as an important substrate determinant. By contrast, we report that Ste24p can cleave both isoprenylated and non-prenylated substrates in vitro, indicating that the isoprenyl moiety is not required for substrate recognition. Steady-state enzyme kinetics are significantly different for prenylated versus nonprenylated substrates, strongly suggestive of a role for substratemembrane interaction in protease function. Mass spectroscopy analyses identify a cleavage preference at bonds where P1 is aliphatic in both isoprenylated and non-prenylated substrates, although this is not necessarily predictive. The identified cleavage sites are not at a fixed distance position relative to the C terminus. In this study, the substrates cleaved by Ste24p are based on known isoprenylated proteins (i.e. K-Ras4b and the yeast a-factor mating pheromone) and non-prenylated biological peptides (A␤ and insulin chains) that are known substrates of the M16A family of soluble zinc-dependent metalloproteases. These results establish that the substrate profile of Ste24p is broader than anticipated, being more similar to that of the M16A protease family than that of the Rce1p CAAX protease with which it has been functionally associated.

Acquisition of accurate data from intramolecular quenched fluorescence protease assays

“…Proteolytic activity of yeast membranes containing overexpressed ZMPSTE24Δcys (cysteine-free human Ste24p, with the five nonconserved cysteines mutated to serine) was characterized with a modified version of existing IQF assays previously utilized for several yeast orthologs of Ste24p [23, 30]. The peptide substrate utilized, Abz-KSKTKC(farnesyl)VIK-Dnp, is cleaved by ZMPSTE24 to yield Abz-KSKTKC(farnesyl) and VIK-Dnp products [23].…”

“…The peptide substrate utilized, Abz-KSKTKC(farnesyl)VIK-Dnp, is cleaved by ZMPSTE24 to yield Abz-KSKTKC(farnesyl) and VIK-Dnp products [23]. To determine the membrane concentration to be used for the assay, a series of two-fold dilutions of ZMPSTE24Δcys enriched membranes were examined in the presence of 1, 10 and 40 μM of Abz-KSKTKC(farnesyl)VIK-Dnp.…”

“…Stock solutions of the peptide substrate and products were prepared by adaptations of published methods [23, 27]. For the substrate Abz-KSKTKC(farnesyl)VIK-Dnp, 26 μL of 100% DMSO was added to a vial containing 1 mg peptide, followed by gradual addition of 974 μL sterile 0.2 μM filtered deionized water in 50 μL increments to yield 1 mL of 1 mg/mL peptide stock in 2.6% v/v DMSO.…”

“…Ste24p is a key factor in several endoplasmic reticulum (ER) processes, including the unfolded protein response, a cellular stress response of the ER [21], and removal of misfolded proteins from the translocon [22]. Also, substrate prenylation has been demonstrated to be dispensable for cleavage by Ste24p [23]. …”

Progeroid syndrome patients with ZMPSTE24 deficiency could benefit when treated with rapamycin and dimethylsulfoxide

“…A recent report now shows that Ste24, a yeast homolog of ZMPSTE24, could cleave both prenylated and nonprenylated substrates, further indicating a much broader substrate profile for Ste24 and possibly ZMPSTE24. From this perspective, our unbiased proteomic study provides additional protein substrates for further studies (Hildebrandt et al 2016)…”