encodes a nonessential SEC24
homologue that interacts in synthetic lethal combinations with other SEC
genes known to be required for COPII-mediated vesicle budding from the ER (Roberg et al. 1999
). Previous work showed that the deletion of LST1
causes the accumulation of the plasma membrane ATPase (Pma1p) in the ER membrane (Roberg et al. 1999
). Thus, Roberg et al. 1999
suggested a role for Lst1p in COPII vesicle budding and Pma1p sorting. Evidence showing a direct role for Lst1p in packaging cargo proteins at the ER required a biochemical approach. To investigate the mechanism of Pma1p packaging and the involvement of Lst1p in this process, we employed a cell-free vesicle budding reaction (Rexach and Schekman 1991
). This reaction allowed us to study the interplay between Lst1p and Sec24p in packaging cargo into vesicles and forming a coat. We found that Sec23/Lst1p is involved in Pma1p recruitment into COPII-coated vesicles.
In contrast to the prediction that Sec23/Lst1p may replace Sec23/Sec24p in the packaging of Pma1p, we found that Sec23/Lst1p functions poorly as a substitute for the normal heterodimer. Instead, we found that both heterodimers are required for efficient packaging of Pma1p into COPII vesicles. Vesicles formed in the presence of the Sec24p and Lst1p heterodimers were similar in buoyant density and morphological appearance, but were ~15% larger in diameter than those formed in a standard incubation. Independent approaches using immunogold staining and chemical cross-linking, followed by denaturing immunoprecipitation with monospecific polyclonal antibodies showed that the two heterodimers copolymerize on a common vesicle species.
Because LST1 is not an essential gene, whereas PMA1 is essential, one would expect traffic of Pma1p not to depend solely on Lst1p. Indeed, we found that high levels of Sec23/Sec24p obviated the requirement for Sec23/Lst1p in our packaging reaction, whereas the converse was not true. At low pH, where Pma1p must function more vigorously, lst1-null cells die, whereas LST1 cells continue to grow. The normal level of Sec23/Sec24p may not suffice in mutant cells grown at low pH.
The Lst1p complex is necessary, but not sufficient for the reconstitution of efficient Pma1p packaging from Lst1-deprived donor membranes (see and ). However, when the cytosol was supplemented with high levels of purified Lst1p complex, a drop in the packaging efficiency of both Pma1p and gpαF was observed (). Because the Lst1p and Sec24p heterodimers cooperate in coat formation, an imbalance of the two may interfere with vesicle budding. This view is supported by the physiologic observation that overexpression of LST1
impairs the growth of wild-type yeast, but cooverexpression of SEC24
compensates for this defect (Roberg et al. 1999
is another nonessential SEC24
homologue of 55% identity (Kurihara et al. 2000
). A Sec23/Iss1p heterodimer replaces Sec23/Sec24p to nearly 50% efficiency in a standard budding assay measuring the packaging of gpαF (Kurihara et al. 2000
). No growth phenotype has yet been ascribed to the iss1
null and, as a result, no uniquely Iss1p dependent cargo has been identified. However, the existence of the homologues and the selective role of Lst1p suggests that distinct Sec24p species may cooperate to increase the range of cargo that can be accommodated in a mixed COPII vesicle. Because they are cytosolic proteins, the Sec24p homologues are easily recruited as needed to mediate the packaging of membrane cargo proteins by direct and most likely signal-mediated interaction.
Coimmunoprecipitation experiments showed that Pma1p associated, directly or indirectly, with Sec23/Sec24p and Sec23/Lst1p. The interaction between Pma1p and Sec23/Sec24p was enhanced when Sec23/Lst1p was also present (). One possibility is that Lst1p makes a direct contact with a cytoplasmically exposed peptide signal on Pma1p. Sec24p may bind weakly to this signal. Further, we suggest that the Lst1p heterodimer is incapable of satisfying the role of the Sec24p heterodimer in COP II coat polymerization. Thus, Lst1p serves as an adaptor to engage Pma1p in the polymerization of a coat achieved by the normal COPII subunits.
Ideally, the Pma1p sorting signal resides within either one of two cytoplasmically oriented loops or termini providing direct access to Lst1p. It seems unlikely that either the NH2
-terminal 27 residues or the COOH-terminal 18 residues is responsible for transport because deletions produce normal steady state levels of plasma membrane Pma1p (Portillo et al. 1989
). Analysis of these mutants in our budding reaction revealed normal packaging efficiencies (not shown). Detailed mutagenesis analysis will be required to identify this signal. However, certain heterologous PMA1
gene expression experiments are instructive. Tobacco PMA1
complements a yeast pma1
mutation, whereas three Arabidopsis
) produce active enzymes which largely remain in the ER (Palmgren and Christensen 1994
; Dexaerde et al. 1995
). The Arabidopsis
homologues may contain a divergent sorting signal.
Do other cargo molecules depend on Lst1p for efficient transport out of the ER? Peng and colleagues observed a slight delay in the maturation of the glycolipid-anchored plasma membrane protein Gas1p in lst1
-null cells (which they term Δsfb3
) (Peng et al. 2000
). However, we have previously shown that Gas1p is similarly packaged when either cytosol or normal purified COPII components are used in vesicle budding reactions (Doering and Schekman 1996
). Pagano et al. 1999
found that several major proteins secreted into the growth medium are missing in culture supernatants from lst1
-null cells (they call this gene SEC24C
). These secretory proteins may derive from membrane-bound precursors that make direct contact with Lst1p in the ER, or they may be carried out of the ER in contact with Pma1p or with recycling membrane receptor proteins that are sorted by Lst1p. Alternatively, their production or secretion may depend on a pH gradient generated by Pma1p across the plasma membrane. The identity of these secreted proteins will help distinguish these possibilities.
Other membrane cargo proteins, such as the general amino acid permease (Gap1p), are recognized and packaged by the standard COPII subunits (Kuehn et al. 1996
). We suggest that such standard passenger proteins are recognized directly or indirectly by Sec24p. Similar considerations apply to anterograde v-SNARE proteins (Springer and Schekman 1998
) and to mammalian membrane proteins, such as the VSV-G protein (Aridor et al. 1998
Sec24p and its homologues may exert a regulatory influence on the Sar1p-selective GAP (GTPase activating protein) activity of Sec23p (Yoshihisa et al. 1993
). In the cycle of COPII vesicle formation, GTP hydrolysis serves to promote coat disassembly, which must be completed before the vesicle can dock and fuse with a target membrane. Although the Sec24p subunit of the heterodimer has no direct influence on the GAP activity of Sec23p, the existence of homologues, at least one of which confers some membrane cargo protein selectivity, suggests that cargo recognition by Sec24p could influence GTP hydrolysis linked to the protein sorting event.
A similar situation may apply in the sorting of cargo by the COPI coat. Coatomer, the assembly protomer of the COPI coat, assembles onto Golgi membranes in the presence of the GTPase, Arf, and GTP (Orci et al. 1993
). Arf GAP and Arf nucleotide exchange activities influence the recruitment of Golgi membrane proteins, such as Erd2p and SNAREs (Aoe et al. 1998
; Spang and Schekman 1998
). Recently, Goldberg 1999
, Goldberg 2000
has shown that coatomer stimulates the activity of Arf GAP and that certain cargo proteins retard the coatomer-stimulated rate of GTP hydrolysis. He suggests a kinetic proofreading model in which preferred cargo proteins delay GTP hydrolysis so as to favor the formation of a tight coatomer substrate complex. Likewise, Sec24p and its homologues may influence Sec23p GAP activity in response to preferred substrates: Gap1p for Sec24p and Pma1p for Lst1p.
Although our evidence is most consistent with a sorting signal on Pma1p decoded by the Lst1p subunit of the COP II coat, the issue of positive sorting vs. transport by default remains unresolved. Most recently, Martinez-Menarguez et al. 1999
resurrected the bulk flow hypothesis with the observation that two abundant secretory proteins in the pancreatic acinar cell, amylase and chymotrypsinogen, are not concentrated in COPII buds or vesicles leaving the ER, but are concentrated in the intermediate compartment by COPI-mediated retrieval of nonsecretory material. In interpreting the Martinez-Menarguez et al. 1999
data, Warren and Mellman 1999
suggested that abundant secretory or membrane cargo proteins may simply exceed the availability of any stoichiometric sorting receptors and therefore such molecules could only be packaged at their prevailing concentrations. They suggested that much less abundant proteins may be more selectively packaged to allow their rapid transport out of the ER. However, for membrane proteins that may be in direct contact with coat subunits, we suggest that even abundant cargo molecules could become concentrated within COPII buds and vesicles. Indeed, Pma1p comprises up to 50% of the total protein of the yeast plasma membrane (van der Rest et al. 1995
It is possible to erect a model reconciling the bulk flow hypothesis and the role of Lst1p in sorting of Pma1p into COPII vesicles. Depending on the subunit structure of Pma1p as it assembles in the ER membrane, normal COPII vesicles may be too small or too sharply curved to admit an intact Pma1p oligomer. Pma1p has been crystallized as a hexameric ring with an outer diameter of 165 Å (Auer et al. 1998
) and preliminary biosynthetic studies suggest an ER oligomer of Pma1p as large as a dodecamer (Lee, M., and R. Sheckman, unpublished observations). Furthermore, Bagnat et al. 2000
, have demonstrated that Pma1p assembles in a glycolipid raft before its export from the ER. This structure may simply not fit in a standard COPII vesicle. Accordingly, we suggest as an alternative to the sorting signal model that the Sec23/Lst1p heterodimer may alter the geometry of COPII coat polymerization creating a vesicle that can accommodate the Pma1p oligomer. Conceivably, the ratio of Sec24p and Lst1p heterodimers in a COPII coat may be regulated to adapt the budding reaction to small or large cargo. In the extreme, large lipoprotein particles and collagen fibers must be transported out of the ER (Schekman and Mellman 1997
), and this transport could require the mammalian equivalent of Lst1p.