Synthetic genetic interactions with temperature-sensitive clathrin Saccharomyces cerevisiae: Roles for synaptojanin-like Inp53p and dynamin-related Vps1p in clathrin-dependent protein sorting at the trans-Golgi network
Es. Bensen et al., Synthetic genetic interactions with temperature-sensitive clathrin Saccharomyces cerevisiae: Roles for synaptojanin-like Inp53p and dynamin-related Vps1p in clathrin-dependent protein sorting at the trans-Golgi network, GENETICS, 154(1), 2000, pp. 83-97
Clathrin is involved in selective protein transport at the Golgi apparatus
and the plasma membrane. To further understand the molecular mechanisms und
erlying clathrin-mediated protein transport pathways, we initiated a geneti
c screen for mutations that display synthetic growth defects when combined
with a temperature-sensitive allele of the clathrin heavy chain gene (chc1-
521) in Saccharomyces cerevisiae. Mutations, when present in cells with wil
d-type clathrin, were analyzed for effects on mating pheromone alpha-factor
s precursor maturation and sorting of die vacuolar protein carboxypeptidase
Y as measures of protein sorting at the yeast trans-Golgi network (TGN) co
mpartment. By these criteria, two classes of mutants were obtained, those w
ith and those without defects in protein sorting at the TGN. One mutant wit
h unaltered protein sorting at the TGN contains a mutation in PTC1, a type
2c serine/threonine phosphatase with widespread influences. The collection
of mutants displaying TGN sorting defects includes members with mutations i
n previously identified vacuolar protein sorting genes (VPS), including the
dynamin family member VPS1. Striking genetic interactions were observed by
combining temperature-sensitive alleles of CHC1 and VPS1, supporting the m
odel that Vps1p is involved in clathrin-mediated vesicle formation at the T
GN. Also in the spectrum of mutants with TGN sorting defects are isolates w
ith mutations in the following: RIC1, encoding a product originally propose
d to participate in ribosome biogenesis; LUV1, encoding a product potential
ly involved in vacuole and microtubule organization; and INP53, encoding a
synaptojanin-like inositol polyphosphate 5-phosphatase. Disruption of INP53
, but not the related INP51 and INP52 genes, resulted in a-factor maturatio
n defects and exacerbated a-factor maturation defects when combined with ch
c1-521. Our findings implicate a wide variety of proteins in clathrin-depen
dent processes and provide evidence for the selective involvement of Inp53p
in clathrin-mediated protein sorting at the TGN.