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J. Cell Biol.,
Volume 142, Number 3, August 10, 1998 635-649
Department of Cell Biology and Anatomy, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
Maturation of the Saccharomyces cerevisiae
a-factor precursor involves COOH-terminal CAAX
processing (prenylation, AAX tripeptide proteolysis,
and carboxyl methylation) followed by cleavage of an
NH2-terminal extension (two sequential proteolytic processing steps). The aim of this study is to clarify the
precise role of Ste24p, a membrane-spanning zinc metalloprotease, in the proteolytic processing of the a-factor precursor. We demonstrated previously that Ste24p
is necessary for the first NH2-terminal processing step
by analysis of radiolabeled a-factor intermediates in
vivo (Fujimura-Kamada, K., F.J. Nouvet, and S. Michaelis. 1997. J. Cell Biol. 136:271-285). In contrast,
using an in vitro protease assay, others showed that
Ste24p (Afc1p) and another gene product, Rce1p,
share partial overlapping function as COOH-terminal
CAAX proteases (Boyartchuk, V.L., M.N. Ashby, and
J. Rine. 1997. Science. 275:1796-1800). Here we resolve
these apparently conflicting results and provide compelling in vivo evidence that Ste24p indeed functions at
two steps of a-factor maturation using two methods. First, direct analysis of a-factor biosynthetic intermediates in the double mutant (ste24
rce1) reveals a previously undetected species (P0*) that fails to be COOH
terminally processed, consistent with redundant roles
for Ste24p and Rce1p in COOH-terminal CAAX processing. Whereas a-factor maturation appears relatively normal in the rce1 single mutant, the ste24 single
mutant accumulates an intermediate that is correctly
COOH terminally processed but is defective in cleavage of the NH2-terminal extension, demonstrating that
Ste24p is also involved in NH2-terminal processing. Together, these data indicate dual roles for Ste24p and a
single role for Rce1p in a-factor processing. Second, by
using a novel set of ubiquitin-a-factor fusions to separate the NH2- and COOH-terminal processing events
of a-factor maturation, we provide independent evidence for the dual roles of Ste24p. We also report here
the isolation of the human (Hs) Ste24p homologue, representing the first human CAAX protease to be
cloned. We show that Hs Ste24p complements the mating defect of the yeast double mutant (ste24 rce1)
strain, implying that like yeast Ste24p, Hs Ste24p can
mediate multiple types of proteolytic events.
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