(Received for publication, August 15, 1995; and in revised form, October 18, 1995)
From the
Formation of vesicular intermediates in protein transport between the endoplasmic reticulum and the Golgi apparatus involves a mechanism that sorts and packages two classes of molecules into transport vesicles: targeting molecules, which are required for targeting and consumption of vesicular intermediates, and cargo proteins. In order to examine the importance of cargo in this packaging reaction, we developed an in vitro assay that quantifies vesicle formation based on segregation of targeting molecules. Here we document that endoplasmic reticulum devoid of cargo proteins is competent in the formation and release of targeting molecule-containing vesicles in a fashion indistinguishable from its normal counterpart. This observation implies that packaging of cargo proteins may be uncoupled from the recruitment of targeting molecules during vesicle budding from the endoplasmic reticulum. Using the same assay, we demonstrate that the packaging of targeting molecules into vesicles is not dependent on the lumenal chaperone, BiP (Kar2p).
The cytosolic events that lead to the formation and release of
vesicles from endoplasmic reticulum (ER) ()were
reconstituted with the use of an enriched ER fraction and three
purified cytosolic fractions (Sec13/31p, Sec23/24p, and Sar1p) that
comprise a novel coat protein complex, COPII (Salama et al.,
1993; Barlowe et al., 1994). A system comprising these factors
and GTP support the inclusion of cargo proteins such as glycosylated
pro-
-factor (gp
f) into 60-mm vesicles that bud from the ER
(Barlowe et al., 1994). In addition to cargo proteins,
ER-derived vesicles contain synaptobrevin-like proteins such as Bet1p,
Bos1p, and Sec22p (Barlowe et al., 1994; Lian and
Ferro-Novick., 1993; Rexach et al., 1994), the targeting
molecules that function as specificity factors by serving as the
docking ``v-SNARE'' in the consumption of vesicles by the
Golgi apparatus (Rothman, 1994). The absence of ER resident proteins in
these transport vesicles suggests that cargo proteins are faithfully
sorted from other ER lumenal proteins (Rexach et al., 1994).
Thus, the COPII proteins plus GTP are sufficient to provide the
cytosolic information necessary to reproduce an authentic vesicle
budding event.
In contrast to advances in the biochemical dissection
of cytosolic requirements for vesicle transport, the molecular events
within the ER lumen that are responsible for segregating and packaging
targeting and cargo molecules into the vesicles are not well
understood. Only two proteins known to play a role in
post-translocational aspects of secretion are lumenally oriented.
Sec12p is a type II membrane glycoprotein with a C-terminal domain of
approximately 100 residues in the ER lumen. The cytosolic domain
catalyzes nucleotide dissociation on Sar1p which serves to initiate the
budding process (Barlowe and Schekman, 1993). In contrast, the lumenal
domain is dispensable; truncated Sec12p that retains only the
N-terminal and membrane anchor domains is sufficient to sustain normal
yeast cell growth. BiP (Kar2p), in addition to its role in polypeptide
translocation (Vogel et al., 1990; Sanders et al.,
1992), is required in the quality control decision to segregate
incompletely folded polypeptides away from cargo that is ready for
transport (Gething and Sambrook, 1992). However, given the defect in
-factor precursor translocation in kar2 mutant membranes,
it has not been possible to test a role for Kar2p in the packaging of
folded proteins into transport vesicles.
In order to determine if
the sorting and packaging of cargo proteins is an essential prelude to
vesicle budding, we developed an assay that measures the distribution
of targeting molecules rather than that of gpf. By monitoring the
release of Bet1p- and Sec22p-containing vesicles from ER membranes
isolated from cells treated with cycloheximide or carrying a
temperature-sensitive allele of kar2, we demonstrated that
neither the packaging of cargo proteins nor Kar2p function are required
to form COPII vesicles.
Figure 2: Isolation of ER-derived vesicles from an ER fraction prepared from cycloheximide-treated cells. a, immunoblot of an ER fraction (2.5 µg of protein) prepared from untreated (M-) or cycloheximide-treated (M+) cells; and Nycodenz gradient-purified vesicles from untreated (lanes 1 and 2) or cycloheximide-treated (lane 3) ER fractions, after incubation with (lanes 2 and 3) or without (lane 1) purified COPII proteins and GTP. b, silver-stained SDS-polyacrylamide gel electrophoresis of samples 1, 2, and 3 described in panel a.
Figure 1: Budding of Bet1p-containing vesicles from ER fractions is dependent on purified COPII proteins and GTP. Various combinations of Sec13/31p, Sec23/24p, Sar1p, and GTP were tested for their ability to package Bet1p (black bar), Sec61p (hatched bar), and Kar2p (white bar) from ER membranes.
Figure 3: Cargo-depleted ER membranes produce Sec22p-containing vesicles. ER membranes prepared from mock (lanes 1-3) or CHX-treated (lanes 4-6) cells were tested for the packaging of Sec22p into vesicles.
Figure 4: An ER fraction devoid of cargo proteins is competent in the formation of Bet1p-containing vesicles. An ER fraction isolated from cycloheximide-treated cells was tested for the packaging of Bet1p (black bar), Sec61p (hatched bar), and Kar2p (white bar) in the presence of the indicated reagents.
Figure 5: Crude cytosols stimulate Bet1p-containing vesicle budding from cargo-depleted ER fraction. ER membranes prepared from mock treated (columns 1-3) and CHX-treated (columns 4-6) cells were tested for the packaging of Bet1p (black bar) and Sec61p (hatched bar) in the presence of 50 µg of crude cytosols prepared from cells grown under normal conditions (cytosol) or in the presence of CHX (CHX cytosol). Dose-response assays showed that half-maximal budding was achieved with 7 µg of cytosol from untreated cells and 8 µg of cytosol from cycloheximide-treated cells.
Figure 6:
Cargo-depleted ER membranes package
exogenous cargo proteins into vesicles. COPII or crude cytosols were
used in the budding of S-gp
f-containing vesicles from
normal (white bar) or cargo-depleted (black bar) ER
membranes.
Figure 7: Budding of v-SNARE-containing vesicles is independent of Kar2p activity. ER membranes prepared from RSY598 (kar2-159) were tested for the packaging of Sec61p (hatched bars) and Bet1p (black bars) in the presence of COPII or 100 µg of crude cytosol at 20 °C (lanes 1-4) or 30 °C (lanes 5-8).
The role of secretory cargo in membrane traffic between the ER and Golgi apparatus has been difficult to resolve. In the extreme case of vesicular stomatitis virus infection, which shuts down host protein synthesis (Zilberstein et al., 1981), the capsid G protein is transported through the secretory pathway as the sole cargo molecule. In yeast, glycoproteins accumulated in the ER in a sec mutant strain are secreted when cells are returned to a permissive temperature in the presence or absence of an inhibition of protein synthesis (cycloheximide) (Novick and Schekman., 1983). In this case the cargo accumulated in the ER may include or represent structural proteins necessary for the traffic event. In support of this view, small vesicles detected as an intermediate in the transport of proteins from the ER in yeast depend upon protein synthesis to accumulate to morphologically observable levels (Kaiser and Schekman, 1990). However, even in this case, a basal level of vesicle traffic may persist in cycloheximide-treated cells. Unfortunately, in vivo experiments have not addressed the cycling of ER to Golgi vesicle targeting (SNARE) proteins in cells treated with cycloheximide.
We
investigated this problem using a cell-free vesicle budding reaction
where the cargo content of the donor ER membrane may be manipulated and
the role of cargo in the formation of an anterograde transport vesicle
can be distinguished from other steps in the ER to Golgi limb of the
secretory pathway. The yeast SNARE proteins Bet1p and Sec22p continue
to be packaged into transport vesicles irrespective of the presence of
cargo molecules. SNARE protein packaging requires the full set of Sec
proteins that comprise the COPII coat shown previously to envelope a
model cargo protein, gpf. No requirement for cargo was detected
when this assay was conducted with ER membranes from
cycloheximide-treated cells incubated with COPII proteins or cytosol
from exponentially growing or cycloheximide-treated cells.
Nevertheless, we cannot rule out the possibility that cells possess a
feedback mechanism that we have not reproduced in vitro. No
requirement for the lumenal chaperone Kar2p was detected when the
packaging of SNARE molecules was assessed using kar2 mutant
membranes incubated with COPII proteins at permissive or restrictive
temperatures. Furthermore, we observed no influence of cargo or Kar2p
on the segregation of SNARE and resident ER membrane proteins that
accompanies the formation of transport vesicles.
We conclude that the availability and packaging of cargo proteins is not essential to the formation of ER-derived vesicles. A similar phenomenon was first discovered in endocytosis of low density lipoprotein receptors in human fibroblasts (Anderson et al., 1978) and more recently in the recycling of pheromone receptors in yeast (Davis et al., 1993), in which the receptors are retrieved from the cell surface irrespective of their liganded state. By analogy to the role of clathrin and its adaptors in receptor-mediated endocytosis, we propose that SNARE and certain membrane cargo (or cargo adaptor) molecules possess structural or sequence information that allows their recognition and capture by COPII. These membrane proteins may share a common domain of interaction on a COPII subunit and thus be packaged together by a stochastic process. Ordinarily, the abundance of both types of membrane protein would ensure their coincident packaging into a single vesicle species. Alternatively, SNARE and membrane cargo (or cargo adaptor) molecules may interact with different COPII subunits, or different portions of the same subunit, thus ensuring a coordinate packaging event. In either case, our model predicts that in the absence of one type of membrane protein packaging of the other would continue.