Syntaxin 5 and Membrin are Packaged Specifically by the Mammalian COPII proteins Sec24c and Sec24d
To study the packaging of cargo membrane proteins into mammalian COPII-coated vesicles, we expressed and purified full-length forms of the four human Sec24 isoforms (Sec24a–d), each complexed with Sec23a. These were used in an in vitro budding assay that employed semi-intact cells and pure, recombinant COPII proteins (Mancias and Goldberg, 2007
). In a previous study, we showed that this assay was able to report the isoform-specific packaging of the SNARE protein Sec22 (which is packaged by Sec24a and Sec24b, but not Sec24c or Sec24d [Mancias and Goldberg, 2007
First, we assessed whether any of the four human Sec24 isoforms are capable of packaging the mammalian SNAREs membrin and syntaxin 5. For this, the semi-intact cells were washed to remove endogenous coat proteins and then incubated with Sec23/24 (comprising Sec23a and Sec24a–d), Sec13a/31a, and Sar1a plus GTP (see Materials and Methods
). The extent of packaging into vesicles of membrin and syntaxin 5 was compared with the packaging of the control cargo protein ERGIC-53 and the control ER resident ribophorin, by immunoblotting of the vesicle contents (). All four forms of Sec23/24 generated COPII vesicles, as assessed by the packaging of ERGIC-53. Strikingly, the Sec24c and Sec24d isoforms specifically packaged both membrin and syntaxin 5, whereas Sec24a and Sec24b did not enrich the SNAREs above background levels.
Signal Sequences on Membrin and Syntaxin 5 for Binding the Sec24 c and d Isoforms
Thus, the human Sec24 isoforms can be grouped according to their SNARE specificity. Sec24c and Sec24d specifically package membrin and syntaxin5, whereas Sec24a and Sec24b specifically package Sec22 (Mancias and Goldberg, 2007
). This grouping conforms to the sequence similarity of the structural core (that is, excluding the hypervariable N-terminal regions) of the Sec24 isoforms: the sequences of Sec24a and Sec24b are 75% identical, and Sec24c and Sec24d are 66% identical, whereas Sec24a and Sec24c are only 31% identical.
Conserved IxM Sequences on Membrin and Syntaxin 5 Bind to Sec24c and Sec24d
To probe for direct interactions between the SNARE proteins and the COPII coat subunits, we expressed portions of the cytosolic domains of membrin and syntaxin 5 fused to the N terminus of glutathione S-transferase (GST), then immobilized the fusion proteins on glutathione sepharose beads and probed for binding to Sec24c. Purified Sec24c was diluted with E. coli lysate proteins, and the specificity of the interactions was assessed by the enrichment of the coat protein from the input mixture ().
Membrin contains an N-terminal folded domain (residues 1–110) upstream of the heptad-repeat SNARE motif and transmembrane domain (Mossessova et al., 2003
). To probe the interaction with membrin, we produced two overlapping fragments, one encompassing the N-terminal domain (residues 1–132) and the other the C-terminal SNARE motif (112–186). Somewhat surprisingly, we found that both the N-terminal and C-terminal polypeptides bound to Sec24c (, lanes 4, 5), implying that the membrin transport signal sequence is situated in the region of overlap of the two constructs. To test this, we produced a membrin fragment that encompasses the linker region, residues 107–126, and we observed an interaction with Sec24c, as predicted (, lane 6); the fragment also binds to Sec24d (data not shown). No binding is observed to the Sec23 subunit (data not shown); thus membrin, like almost all other cargo molecules studied to date, binds to the Sec24 subunit of the COPII coat (Lee et al., 2004
To assess the specificity of the interaction and to pinpoint key residues, we performed an alanine scan through the membrin fragment 107–126. (lanes 7–12) shows the effects of mutations through the central region of the membrin sequence. Mutation of two residues, I118A and M1 20A significantly impaired binding to Sec24c, whereas mutation of neighboring residues had no appreciable effect on binding (, lanes 7–12). We refer to this core region of the membrin sequence as the IxM motif; a more definitive test of the role of the IxM motif as a transport signal was possible once we had mapped the interaction site of membrin IxM with Sec24c and Sec24d by X-ray crystallography, and this analysis is described below.
In a previous study on the S. cerevisiae
COPII coat (Mossessova et al., 2003
), we detected no interaction between Sec24 and Bos1 (yeast membrin). Consistent with this, the IxM sequence that we have now identified is not present in the yeast Bos1 polypeptide (). By contrast, the IxM motif is highly conserved both in sequence and position in membrin proteins of higher eukaryotes; in the sequences that we studied, the methionine residue of IxM is invariant, whereas the isoleucine residue can change conservatively to leucine.
Earlier studies on the S. cerevisiae
COPII coat raised a similar puzzle regarding the signals for packaging syntaxin 5 into vesicles (Mossessova et al., 2003
). Specifically, a YNNSNPF sequence on yeast Sed5/syntaxin 5 is the primary signal for ER exit, and a LxxME motif may be a secondary signal (Miller et al., 2005
; Mossessova et al., 2003
); however, as illustrated in (lower panel), the YNNSNPF and LxxME sequences are unique to the S. cerevisiae
SNARE protein. These sequences are located in a linker region of the syntaxin 5 polypeptide that separates the N-terminal regulatory domain (NTD) and the C-terminal SNARE motif and transmembrane domain (Bracher and Weissenhorn, 2002
). Strikingly, when we studied sequences of the syntaxin 5 linker region from higher eukaryotes we identified a highly conserved IxM sequence in a central location ().
To test whether the IxM sequence on syntaxin 5 binds to Sec24c, we produced an appropriate fragment of syntaxin 5 (fused to GST) containing the putative IxM signal (residues 151–220) and tested this for binding to Sec24c and Sec24d. The ~70-residue syntaxin 5 peptide does indeed bind to Sec24c and Sec24d (, lane 4 and data not shown). The syntaxin 5 peptide was shortened to just a 20-residue element (residues 181–200) which also binds to the coat proteins (, lane 5). Point mutations to alanine of a core sequence of the 20-residue peptide had the predicted effect: the mutations I191A and M193A impaired binding to Sec24c (, lanes 8, 10), whereas mutation of neighboring residues had little or no effect on binding ( lanes 6, 7, 9 and 11).
In summary, we have defined interactions between the COPII coat machinery and mammalian membrin and syntaxin 5, involving a highly conserved IxM motif that we infer is a signal for cargo packaging into COPII-coated vesicles. In order to confirm the specificity of the interactions, and to establish a structural framework for functional tests of the IxM sequence as a signal for SNARE packaging, we initiated X-ray crystallographic analysis of the coat–signal interactions.
Molecular Recognition of the IxM Signal by the COPII Machinery
For crystallization experiments we purified truncated versions of the Sec24 proteins that lack the hypervariable, unstructured N-terminal regions (see Materials and Methods
). These were used in crystallization trials of Sec23/24c–d heterodimers and Sec24c–d monomers, and yielded two new crystal forms for the Sec24c and Sec23a/24d complexes. Synthetic membrin and syntaxin 5 peptides were soaked into fully-grown crystals at high concentrations (4 mM), and the co-crystal structures of Sec23a/24d•membrin and Sec23a/24d•syntaxin 5 were determined by standard X-ray crystallographic methods. In both cases, peptide sequences could be identified by difference-Fourier analysis and were modeled straightforwardly into residual electron density (see Materials and Methods
Data Collection and Refinement Statistics
The IxM-containing peptides of syntaxin 5 and membrin bind to a common site on the equatorial surface of Sec24d (). The binding site is distal to the interface with Sec23; it is roughly equidistant to the A- and B-sites described previously on yeast Sec24 (Mossessova et al., 2003
); and we estimate that the binding site is situated ~10–15 Å from membrane when the COPII coat is bound to the membrane surface.
Molecular Recognition of Syntaxin 5 and Membrin IxM Transport Signals by Sec24d
The co-crystal structure of syntaxin 5 and Sec23a/24d was determined with the syntaxin 5 peptide 188DVAIDMMDP196 bound, and strong electron density is observed for all residues except the C-terminal Asp195 and Pro196 (). The co-crystal structure comprising membrin peptide bound to Sec23a/24d used the membrin sequence 116TTIPMDE122, and in this case electron density is observed for all residues except C-terminal Glu122 (). The syntaxin 5 and membrin peptides bind in very similar conformations, with the IxM motif in a central position. Specifically, the peptides adopt an extended conformation and bind in a β-strand arrangement to a surface loop connecting helices αL and αM (residues 825–841) in the all-helical domain of Sec24 (). The binding site is framed by the most C-terminal α-helix (αS) and the most N-terminal loop-helix in the Sec24d crystal structure. The binding interactions observed between the peptides and Sec24 are consistent with the results of the alanine-scan mutagenesis (). Only the isoleucine and methionine side chains of the IxM motif make extensive interactions with Sec24d. Primary hydrophobic contacts are made between the conserved methionine residue and Sec24d residues Leu834 (on the αL-αM element) and His1025 (on helix αS). For both syntaxin 5 and membrin, the isoleucine residue of the IxM motif makes hydrophobic contacts with residues Leu834, Leu836 and Leu1021 of Sec24d. Although the side chains neighboring the IxM motif do not form contacts with Sec24d, the main-chain atoms of these residues likely contribute to binding, specifically though the antiparallel β-strand hydrogen-bonding arrangement involving the αL-αM element ( and ). Finally, the isoleucine and methionine residues of the IxM motif make intra-peptide hydrophobic contacts that may help stabilize the peptide conformation when bound to Sec24d.
Structural Basis for Signal-Mediated Packaging of Membrin and Syntaxin 5 into COPII vesicles
Packaging of Syntaxin 5 and Membrin by Mammalian COPII Vesicles
The residues lining the IxM-binding site on Sec24c and Sec24d are highly conserved (). Indeed, the key binding-site residues Leu834, Leu836, Leu1021 and His1025 (numbering for Sec24d) are invariant in the Sec24c/d family. Additional residues that may provide stabilization within the αL-αM loop are also conserved, namely Gln833, Ile835, and Pro837.
On the basis of these structural observations, we designed a binding-site triple mutant of Sec24c in order to test the relevance of the co-crystal structure and of the role of the IxM motif in SNARE packaging. Specifically, we mutated the binding-site sequence 895LIL897 on Sec24c (equivalent to 834LIL836 on Sec24d) to 895AAA897; the location of these three side chains is shown in . Vesicles generated with the mutant protein contained substantially lower levels (at or near background) of membrin and syntaxin 5 when compared to that obtained with wild-type protein (). This seems not to be due to a general defect in generating COPII vesicles or in cargo packaging, as the mutant coat protein efficiently packaged ERGIC-53.
In summary, we propose that the interaction between the IxM motif and Sec24c/d is the primary mechanism for packaging membrin and syntaxin 5 into COPII-coated vesicles, and that the IxM motif is a highly conserved signal for ER export of these SNARE proteins. Whereas S. cerevisiae
Sed5 (syntaxin 5) uses YNNSNPF and LxxME signals for ER exit (Miller et al., 2005
; Mossessova et al., 2003
), it seems that IxM is the canonical motif for ER exit of syntaxin 5 and membrin.
Conserved B Site for Binding Bet1 LxxLE and DxE Signal Sequences
It was noted in structural studies of the Sec23/24 complex (Bi et al., 2002
) that the Sec24 B site—which is now known to bind LxxLE and DxE signals—is well conserved across all isoforms of Sec24 (in particular, the B site is highly similar in S. cerevisiae
Sec24 and Lst1, a yeast homolog of Sec24). Nevertheless, in vitro budding experiments show that yeast Sec24 can package Bet1 via its LxxLE motif, whereas Lst1 fails to package Bet1 (Miller et al., 2002
). A structural explanation was suggested by X-ray analysis of the Sec24•Bet1 complex; this indicated that subtle residue changes at the B site were responsible for the differential packaging activity of the Sec24 forms. In particular, residue Leu582 of Sec24, which forms van der Waals contacts with Bet1 LxxLE, is changed to aspartic acid (Mossessova et al., 2003
Interestingly, Leu582 of Sec24, or rather the equivalent residue position in the human proteins, is conserved as a leucine in Sec24a and Sec24b, but is an aspartic acid in Sec24c and Sec24d. On this basis, we tested the prediction that the B-site of human Sec24 isoforms has differential selectivity for LxxLE and DxE signal sequences. This was done via pull-down experiments involving the DxE signal of viral VSV-Glycoprotein. The DxE sequence of VSV-G was the first ER exit signal to be discovered (Nishimura et al., 1999
). More recently, DxE signals have been uncovered in endogenous proteins, in particular yeast Sys1, and this latter protein has been subject to extensive structure/function analysis (Miller et al., 2003
; Mossessova et al., 2003
; Votsmeier and Gallwitz, 2001
The C-terminal tail of VSV-G was produced as a C-terminal fusion to GST and this was used to test for interactions with the human isoforms Sec24a–d (). VSV-G interacted strongly with Sec24a and Sec24b, but not with Sec24c or Sec24d (, lanes 4, 8, 12, 16). Alanine mutations of the DxE motif of VSV-G showed a strict requirement for both the aspartate and glutamate residues of the signal (, lanes 19, 21). By contrast, the central isoleucine residue could be changed to alanine with only minor loss of affinity (, lane 20). In summary, there is indeed selectivity among the human Sec24 isoforms for binding DxE signals.
Molecular Recognition of Bet1 and VSV-G Transport Signals at the B site of Sec24a and Sec24b
Based on these observations, the minimal VSV-G peptide sequence 499
was synthesized. The peptide, which was highly soluble, was soaked into fully-grown crystals of the Sec23a/24a•Sec22b complex. The crystal structure of the ~200 kDa Sec23a/24a•Sec22b complex was determined in a previous study (Mancias and Goldberg, 2007
); importantly, these crystals are grown in a PEG/salt solution that is compatible with peptide binding (see Materials and Methods
), and the B-site is thoroughly exposed to solvent in the crystals and thus available for binding the signal sequence. The crystal structure of Sec23a/24a•Sec22b bound to the VSV-G peptide was refined to 2.7Å resolution (). In the structure, the B site of Sec24a is very similar, in terms of the conformation of key residue side chains, to the B site of yeast Sec24 when bound to DxE and LxxLE signals (Mossessova et al., 2003
). As expected, strong electron density in difference Fourier maps was observed at the B site for a portion of the VSV-G peptide, 502
(). The binding mode is highly similar to the DxE motif of Sys1 bound to yeast Sec24 (Mossessova et al., 2003
). Specifically, the 504
residues of VSV-G adopt a helical configuration and the glutamate side chain makes salt-bridge contacts with Arg752 of Sec24a. Importantly, residue Asp503 of VSV-G adopts a left-handed backbone conformation, thereby allowing the carboxylate group of its side chain to form a bivalent interaction with Arg750 of Sec24a (). The same arrangement—involving a left-handed backbone conformation of the aspartate residue—is observed for Sys1, and is the defining feature of the differential binding modes of DxE- and LxxLE-class signals at the B site (Mossessova et al., 2003
Finally, we note that the VSV-G sequence, 499QIYTDIEMNR508, has an IxM motif nested in the DxE signal. Despite this, the VSV-G signal does not bind to Sec24c or Sec24d (), suggesting that there are residue restrictions at the sites neighboring IxM (possibly including a requirement for a short or β-branched amino acid immediately upstream of the isoleucine residue, needed to stabilize the extended β-stranded conformation of the IxM signal). Further studies are required to explore the detailed sequence requirements for IxM binding, and to test the idea that the nesting of a cryptic IxM signal in the DxE motif of VSV-G renders the 499QIYTDIEMNR508 C-terminal tail of the viral protein more resistant functionally to point mutations.
The LxxLE transport signal of yeast Bet1 is believed to be the primary signal for ER exit of this SNARE protein (Miller et al., 2003
; Mossessova et al., 2003
). Motifs similar to LxxLE are conserved in Bet1 proteins across species, both in terms of sequence and position along the SNARE polypeptide. However, significant sequence variation is observed; thus, human Bet1 bears the motif YxxCE within the sequence 24
at the N terminus of the SNARE motif. Nevertheless, we concluded that human YxxCE is likely to be functionally equivalent to yeast LxxLE, based on an analysis of the yeast Sec24•Bet1 interaction. Specifically, the invariant glutamate residue forms an intricate arrangement of hydrogen-bonding interactions with binding-site residues, whereas the role of the leucine residues is merely to form hydrophobic elements that face Sec24 and that help stabilize the peptide helical conformation (Mossessova et al., 2003
To test whether the YxxCE motif of human Bet1 binds to Sec24a/b in this manner we co-crystallized the peptide with Sec23a/24a•Sec22b. Initially, crystallization studies proved difficult owing to low solubility of the Bet1 sequence 17–33; similar problems had arisen with the yeast Bet1 peptide (Mossessova et al., 2003
). Human Bet1 peptides were sufficiently soluble when N-terminal residues were absent, so the co-crystal structure was determined with the peptide 24
. The binding mode of the peptide is highly similar to that of yeast Bet1 LxxLE bound to Sec24. There are four noteworthy observations. Firstly, residue Glu29 of YxxCE forms salt-bridge contacts with Arg752 of Sec24a (). Secondly, Sec24a recognizes the helical configuration of the signal peptide by capping the main-chain carbonyl oxygen atoms of 29
via the guanidinium groups of arginine residues 430 and 435 (). Thirdly, the cysteine residue of the human Bet1 sequence occupies the same binding site as the second leucine of yeast Bet1 LxxLE. Finally, as in the yeast Sec24•Bet1 complex, N-terminal residues of the bound peptide are mobile in the crystal, such that electron density is absent for residues 24
(see Mossessova et al. 
for a discussion of the possible role of these upstream residues).
This analysis shows that the B site of Sec24 subunits is conserved from yeast to humans for binding DxE and LxxLE ER exit signals, but only among isoforms of the Sec24a/b class of COPII coat proteins.
Diversity of ER Exit Signals and Binding Sites on COPII Proteins
The model in summarizes the results of structural studies on COPII subunit cargo selectivity. The picture includes part of the 60-nm cuboctahedron cage, which is formed from 24 copies of the (Sec13/31)2
assembly unit (Fath et al., 2007
; Stagg et al., 2006
). Each of the 48 copies of Sec31 in the cage can bind to a Sec23/24•Sar1 complex (Bi et al., 2007
), but we have included just two copies of Sec23/24•Sar1 in the picture for clarity.
Arrangement of Cargo-Binding Sites in the COPII Coat
Multiple isoforms of Sec24 expand the range of cargo accommodated in mammalian COPII vesicles, but there is no evidence that Sec24 isoforms are segregated into distinct vesicles; instead, mammalian COPII-coated vesicles may include the full complement of Sec24 isoforms. In we have indicated the binding sites for Sec22, Bet1 and VSV-G signals on a model of Sec24a/b, and the binding site of the membrin and syntaxin 5 IxM motif on Sec24c/d, along with the site occupied by the YNNSNPF signal of Sed5/syntaxin 5 in the yeast system. The manner in which Bet1 and Sec22 are packaged by Sec24a and Sec24b is conserved from yeast to humans. By contrast, the YYNSNPF motif seems to be a S. cerevisiae novelty for packaging syntaxin 5 at a site that has (as yet) no known role in the human Sec24 proteins. The conserved mechanism for packaging syntaxin 5 and membrin employs the IxM motif and COPII subunits of the Sec24c/d class.
Structural Basis for Cargo Discrimination by Human Sec24 Proteins
To complete the structure/function analysis in this study, we sought a structural explanation for the ability of the human Sec24 isoforms to discriminate among membrin, syntaxin 5 and Sec22. To this end, we solved structures of Sec24b and Sec24c, which together with the Sec23a/Sec24d and Sec23a/Sec24a•Sec22 complexes provide a complete set of structures for human Sec24 proteins (see Materials and Methods
Comparison of the crystal structures provides a clear explanation for the selectivity towards the IxM signal of membrin and syntaxin 5. highlights the αL-αM structural element that forms the core of the IxM binding site on Sec24c and Sec24d. Strikingly, although the αL-αM conformation is conserved in Sec24a and Sec24b (), it serves as a binding site not for a signal sequence but for a short β-strand in the N-terminus of the Sec24 polypeptide (residues 349–353 in Sec24a; 523–527 in Sec24b). This self-interaction involves an antiparallel β-strand arrangement similar to the IxM peptide binding to Sec24c/d. Thus, a peptide element is conserved in Sec24a and Sec24b (colored red in ) to bind across the αL-αM site, whereas this element has a distinct amino-acid sequence in Sec24c and Sec24d and adopts an α-helix that forms part of the IxM-binding site (colored red in ). (The polypeptide chains of the four human Sec24 have three regions: an N-terminal hypervariable region that is probably unstructured in solution; an adjacent short stretch (colored red in ) which modulates IxM signal binding; and the “core” sequence that starts from the β-barrel domain [Bi et al., 2002
] and is conserved in all four isoforms).
Structural Basis for Cargo Discrimination by Human Sec24 Isoforms
Finally, we compared the structures of Sec24c and Sec24d determined in this study with the structure of Sec23a/Sec24a•Sec22b determined previously (Mancias and Goldberg, 2007
), to provide an explanation for Sec24 isoform specificity for the SNARE Sec22. As shown in , the conformation of the Sec22-binding site is similar in Sec24a and Sec24c (and also in Sec24b and Sec24d, not shown). However, key residue changes preclude Sec22 binding to Sec24c/d. Several of these are indicated in , for example Sec24a residue Pro500 changes to phenylalanine in Sec24c, but the most important change seems to be to Sec24a residue Arg541. This arginine residue was pinpointed originally in a genetic screen for dysfunctional S. cerevisiae
Sec24 proteins; it is essential for tight binding of Sec22 to Sec23a/24a, and for packaging into COPII-coated vesicles (Mancias and Goldberg, 2007
; Miller et al., 2003
). The Sec24a mutant R541A is unable to package Sec22b (as is the Sec24b mutant R715A). Strikingly, the equivalent position in Sec24c harbors an alanine residue ().
Thus, we have described two distinct mechanisms for controlling the specificity of Sec24 proteins for cargo: in the cases of Sec22 and LxxLE/DxE signals, there is a straightforward divergence of binding-site residues; in the case of the IxM signal, a more elaborate mechanism entails the occlusion of the IxM-binding site by an N-terminal polypeptide element of the Sec24a/b subunits.
The two classes of Sec24 subunits, Sec24a/b and Sec24c/d, have non-overlapping selectivity for the ER exit signals analyzed in this study, and we predict that this trend will continue for the numerous signals that probably remain to be discovered. To date, only one interaction partner is shared by all four Sec24 subunits, namely Sec23 (Fromme et al., 2008