4 research outputs found
Functional characterization of the yeast ER packaging chaperone Shr3p
All living cells are enclosed by a plasma membrane (PM). The PM is a
dynamic structure comprised of lipids and proteins that establishes and
maintains the integrity of the cell. Cells utilize the PM to regulate
metabolism by selectively altering the flow of metabolites entering the
cell. The integral protein components of the PM, e.g., metabolite
transport systems, are co- translationally inserted into the membrane of
the endoplasmic reticulum (ER). Subsequent to membrane insertion, these
proteins must fold properly to attain native conformations, a process
that often requires specific processing events. At an early stage in the
secretory pathway, integral PM and secreted proteins are transported from
the ER to the Golgi via ER-derived transport vesicles. In vitro studies
have shown that a set of cytosolic proteins (Sar1p, Sec23p/Sec24p
complex, and Sec13p/Sec31p complex) coordinately function to catalyze the
formation of ER transport vesicles. These components are recruited to the
ER membrane and subsequently oligomerize to form a vesicle coat structure
known as COPII Cargo proteins are separated from resident ER proteins
concomitantly with COPII vesicle formation.
Amino acid uptake in yeast is mediated by general and specific amino acid
permeases (aaps). Complete sequencing of the yeast genome has revealed
the existence of 18 aaps that share extensive sequence homology. These
permeases are integral polytopic membrane proteins comprised of twelve
hydrophobic domains. Aaps require Shr3p, a 23 kDa membrane component of
the ER, to be functionally expressed. In cells lacking Shr3p, aaps
accumulate in the ER and are not transported to the plasma membrane. The
ER export block observed in shr3 null mutants is specific for permeases,
other plasma membrane proteins, secretory proteins and vacuolar proteins
are processed and targeted correctly.
This thesis documents experiments that examine the molecular basis of
Shr3p function. The membrane topology and folding of the general aap
(GAP]) in SHR3 and shr3alpha null mutant cells was determined. No
detectable differences in the orientation of transmembrane spanning
domains was found, and shr3alpha null mutants do not express enhanced
levels of ER stress response proteins even when aap expression is
derepressed. These findings indicate that the folding and membrane
topology of Gap1p is normal and thus independent of Shr3p function. Shr3p
physically associates with Gap1p, but not with other polytopic membrane
proteins such as Sec61p, Ga12p or Pma1p, in a transient complex that can
be purified from N-dodecylmaltoside solubilized membranes. The COPII
coatomer components See 1 3p, Sec23p, Sec24p, and Sec3 1p, ut not Sar1p,
bind Shr3p via interactions with its carboxy-terminal domain. By
facilitating the membrane association of COPII coatomer components in
close association with aaps, Shr3p is likely to function as a primer of
ERderived transport vesicle formation that directs the assembly of
vesicle buds around aaps.
In SHR3 wild-type cells the degradation of Gap1p and the histidine
specific permease (Hip1p) is dependent upon PEP4, demonstrating that in
these cells aaps exit the ER and are degraded in the vacuole. In
shr3alpha cells however, Gap1p and Hip1p are more stable and their
degradation occurs independently of PEP4. Instead, in the absence of
Shr3p, the degradation of Gap1p and Hip1p is dependent on UBC7, a gene
that encodes an E2 ubiquitin conjugating enzyme required for proper ER
associated degradation (ERAD). The degradation of well characterized ERAD
substrates CPY* and Pdr5 * was decreased in shr3 null mutants, suggesting
that the accumulation of aaps in the ER saturates a limiting ERAD
component. In contrast the degradation rate of Fur4*, a mutant version of
the uracil permease, was not affected in shr3 null mutants. Additionally,
Hip1p was significantly more stable than Gap1p in cells lacking
Hrd1p/Der3p, the ER localized membrane bound E3 ubiquitin protein-ligase.
These latter results demonstrate that despite extensive sequence and
structural similarities, and common requirement for Shr3p, different aaps
are degraded by alternative ERAD dependent processes