-tagged full-length hamster Sar1 was expressed and purified from Escherichia coli
(Rowe et al., 1996
), but failed to crystallize due to formation of higher order oligomers. Therefore, we generated an NH2
-terminal Δ9-Sar1A mutant that was monodispersed in solution. The protein had a native apparent molecular mass of 43.6 kD corresponding to a dimer that yielded diffraction quality crystals (unpublished data). The three-dimensional structure of Δ9-Sar1 in the GDP-bound form (abbreviated as Sar1-GDP) was determined by the multiwavelength anomalous diffraction (MAD) method using data from its selenomethionine derivative to 2.0 Å resolution. Sar1 is sufficiently different than Arf GTPases, such that obtaining a structure based on homology modeling with Arf was difficult.
The structure was refined against native Sar1-GDP data to 1.7 Å to Rcryst
values of 22.2 and 24.2%, respectively ( and II; )
. Overall, the structure was well defined with an average B value of 21.4 Å2
, and an estimated overall coordinate error of 0.22 Å. Consistent with dynamic light scattering measurements in aqueous solution, two Sar1-GDP molecules (A and B) formed a dimer in the crystallographic asymmetric unit with the noncrystallographic twofold axis running between and parallel to the β2 strands ( A). The root mean square deviation (rmsd) of 0.56 Å for 166 equivalent Cα atoms confirmed highly similar core structures. However, the dimerization through the parallel pairing of segments of their respective β2 strands (molecule A, 61-SEELTI-66; molecule B, 60-TSEELT-65) is slightly asymmetric and is mediated by five main chain hydrogen bonds and three side chain hydrogen bonds (63E[molecule A]–46K[molecule B]; 46K[A]–61S[B]; and 60T[A]–81Q[B]). This dimerization differs from Arf1-GDP, which forms a symmetric dimer through its unique β2ε strands (Amor et al., 1994
; Greasley et al., 1995
Statistics of x-ray data collection and structural refinement for Sar1-GDP crystals
The structural core of Sar1
Sar1 ( C) has a structural core of six central β strands (5 parallel, 1 antiparallel [β2]; in order: 6, 5, 4, 1, 3, 2) in a relatively flat β sheet that is sandwiched between three α helices on either side. This motif forms a typical Ras superfamily guanine nucleotide-binding pocket (Schweins and Wittinghofer, 1994
), with the relatively flat β sheet being more characteristic, but different than that of the Arf family GTPases (Amor et al., 1994
; Greasley et al., 1995
) (Fig. S1, available at http://www.jcb.org/cgi/content/full/200106039/DC1
). The nucleotide (GDP) is partially buried in the binding pocket with the ribose ring more exposed than the phosphate and purine moieties ( C). The hydrophobic face of the purine ring is sandwiched by the aliphatic side chains of Leu181 and Lys135.
The GDP-binding site includes residues found in all of the highly conserved binding motifs diagnostic of GTPases. Thr39 is found in the conserved GxxxxGKT39
motif, and is essential for Sar1 function. In particular, the Sar1 dominant negative mutant Sar1[T39N] is a potent inhibitor of COPII vesicle formation (Kuge et al., 1994
; Aridor and Balch, 1996
; Rowe et al., 1996
). Other Ras superfamily GTPases with mutations in the homologous residue are restricted to their GDP-bound form and inhibit their respective GEF activity. We have shown that Sar1[T39N] inhibits wild-type Sar1 function in vivo and in vitro by interfering with its interaction with mammalian Sec12 (mSec12), the ER-associated GEF that is required for Sar1 activation and COPII vesicle formation (Weissman et al., 2001
). Moreover, Asp34 within the GxD34
xxGK(S/T) guanine nucleotide binding motif is an invariant residue in the Sar1 family that aligns with Gly12 of Ras. Mutation of Gly12 in Ras greatly reduces the intrinsic GTP hydrolysis rate leading to prolonged activation and cell transformation (Wittinghofer and Pai, 1991
). In contrast, mutation of Asp34 to Gly in yeast Sar1 interferes with GTP-loading in vitro and destroys its ability to function in vesicle budding (Saito et al., 1998
The Mg ion involved in guanine nucleotide binding in molecule B of Sar1-GDP shows octahedral coordination geometry typical for small GTPases. It is coordinated by an oxygen atom of the β phosphate of GDP, the hydroxyl oxygen of a conserved Thr residue (residue 39 in Sar1-GDP) of the phosphate-binding GxxxxGKS/T motif ( C) (corresponding to residues 10–17 of Ras where x is a variable residue) (Wittinghofer and Pai, 1991
), and four water molecules with Mg-O bond distances of 2.23 Å, 2.19 Å, 2.31 Å, 2.31 Å, 2.30 Å, and 2.32 Å, respectively. Two of the water molecules (w22 and w5) are further hydrogen bonded to Asp75 that belongs to the highly conserved DxxG motif in the switch II region.
Organization of switch regions in Sar1-GDP
Ras superfamily GTPases contain two switch regions (I and II) that are involved in interaction with effectors that promote guanine nucleotide exchange and hydrolysis (Vetter and Wittinghofer, 2001
). In Sar1, both switch I and II loops are proximal to the dimer interface ( A). Based on structural alignment with other Ras superfamily GTPases, the switch regions in Sar1 encompass residues 48–59 (switch I) and 78–94 (switch II). The Sar1 switch I residues (55–59) in both molecules are close to the guanine nucleotide, consistent with the role of the switch I loop in mediating indirect interaction between effector molecules and guanine nucleotide. In contrast, a segment of the switch I residues in Arf1-GDP form a novel β2ε strand that is distant from the guanine nucleotide binding site (Amor et al., 1994
; Greasley et al., 1995
). Consistent with the role of the two switch regions in mediating Sar1 function, mutation of specific residues in either switch I (Leu57) ( C) or II (Lys87) ( C) interferes with COPII vesicle budding and ER export in vivo and in vitro (unpublished data). The homologous mutations in ARF1 disrupt the interaction of ARF1 with the ARF1-specific GEF ARNO (Beraud-Dufour et al., 1998
Residues 10–12, 49–54, and 79–81 of molecule A, and residues 10–12 and 50–54 of molecule B that include segments in the switch regions, lack electron density and were omitted from final model of Sar1 (). A similar lack of density in the switch regions of other GDP-bound forms of Ras superfamily members is frequently observed and reflects the extensive conformational changes that accompany interaction with effectors and GDP/GTP exchange. Whereas switch II (79–82) is well ordered in molecule B, the disorder in molecule A is partly due to the loss of a hydrogen bond between His79 and Asp34, which results in Asp34 swinging away from His79. His79 in Sar1 corresponds to Gln61 in Ras-GTP (Wittinghofer and Pai, 1991
) and Gln71 in Arf1-GTP (Amor et al., 1994
; Greasley et al., 1995
), both of which are important for the hydrolysis of GTP. Consistent with this role, the Sarl[H79G] mutant is a potent inhibitor of ER to Golgi transport that prevents the disassembly of COPII vesicle coats as a consequence of its reduced rate of Sec23/24 GAP-stimulated hydrolysis (Rowe et al., 1996
; Aridor et al., 1998
Distinctive structural features of Sar1
Sar1 has a number of distinctive features that separate it from Ras superfamily GTPases including Arf (Fig. S1, available at http://www.jcb.org/cgi/content/full/200106039/DC1
). Sar1-GDP lacks the extended COOH terminus found in Ras, Rho/Rac/Cdc42, and Rab GTPases. Instead, the COOH-terminal residues of Sar1-GDP are highly ordered, as in Arf1 and Arf6 (Amor et al., 1994
; Greasley et al., 1995
; Menetrey et al., 2000
), and form an amphipathic helix (α5; residues 187–197) that covers the hydrophobic core of the central β sheet (). Other features differentiate Sar1 from Arf1. First, as indicated above, Sar1-GDP lacks the extra β strand (β2ε) found in Arf1 (Amor et al., 1994
; Greasley et al., 1995
) and Arf6 (Menetrey et al., 2000
), which is adjacent to the β2 strand in the switch I region. The β2ε strand plays a critical role in transition from the GDP- to the GTP-bound state after interaction with Arf-specific GEFs (Antonny et al., 1997
; Paris et al., 1997
; Beraud-Dufour et al., 1999
A second unique structural feature of Sar1 not observed in Arf GTPases or other Ras superfamily members is a novel insert encompassing residues 156–171, which are quite variable in sequence among evolutionarily distant members of the Sar1 family. These residues form a long surface-exposed loop (denoted as the Ω loop, as their NH2
and COOH termini are adjacent to each other) connecting helix α4 and strand β6 ( C). The insert loop has a relatively high B value, 36 Å2
as compared with the overall value of 21 Å2
. As β sheet proteins have a tendency to associate with other β sheet proteins through the pairing of their edge β strands, the high B value and the fact that the Sar1 Ω loop is located at the edge of the core β sheet suggests a potential functional role in regulating Sar1 interactions with other components of the COPII machinery. To test this possibility, we mutated the highly conserved Thr158 to Ala and analyzed its ability to support export of vesicular stomatitis virus glycoprotein (VSV-G) from the ER using an assay that reconstitutes COPII vesicle budding from ER microsomes in vitro (Rowe et al., 1996
). Here, VSV-G–containing ER microsomes are incubated in the presence of Sar1(wild type and mutant) and purified Sec23/24 and Sec13/31 components, and the amount of VSV-G cargo recovered in the COPII vesicle fraction is quantitated. Consistent with the importance of this region in Sar1 function, we observed a complete loss activity with the Sar1[T158A] mutant (
Figure 2. The α1′ helix is critical for recognizing the Sec23/24 GAP complex. (A) ER microsomes and purified, recombinant Sar1 wild-type and mutant proteins were prepared as described (Rowe and Balch, 1995). COPII vesicle budding assays were performed (more ...)
Sar1 contains an NH2-terminal α1' helix domain that facilitates interaction with the Sec23/24 GAP complex
-terminal residues (15-VLNFL-19) of Sar1-GDP form an amphipathic helix (α1′) ( C, green). Arf1 GTPases also contain an NH2
-terminal α1′ helix (Amor et al., 1994
; Greasley et al., 1995
) that is essential for function, is believed to facilitate binding of Arf1 to the lipid bilayer (Antonny et al., 1997
; Beraud-Dufour et al., 1999
), and is essential for interaction with phospholipase D (Jones et al., 1999
). However, the orientation of the Sar1 α1′ helix is very different than that of Arf1. Whereas the NH2
-terminal helices of Arf1-GDP and Arf6-GDP run parallel to the β strands of the central β sheet (Amor et al., 1994
; Greasley et al., 1995
; Menetrey et al., 2000
), the Sar1-GDP NH2
-terminal helix is oriented perpendicular to the β sheet ( C). The NH2
-terminal helix is not found in other Ras superfamily GTPases, although a very short helix linked to an extended NH2
-terminal loop is found in the GDP structure of Arl3, an Arf-like GTPase of unknown function (Hillig et al., 2000
To explore the potential role of the α1′-helix in Sar1 function, we generated a mutant in which the hydrophobic residues Val 15, Leu 16, Phe 18, and Leu 19 that principally contribute to packing of the α1′ helix to the hydrophobic core ( C), were mutated to Ala (Sar1[AAAA]). We first tested the ability of the mutant to support export of VSV-G from the ER into COPII vesicles in vitro (Rowe et al., 1996
). Compared with wild-type Sar1 which supports efficient export from the ER, Sar1[AAAA] lost all activity ( A). Loss of activity was not due to misfolding of the core fold of Sar1, because the [AAAA] substitution had no effect on nucleotide binding (Fig. S2 A, available at http://www.jcb.org/cgi/content/full/200106039/DC1
). Thus, the NH2
-terminal α1′ helix is essential for Sar1 function.
We next examined whether the Sar1[AAAA] mutant could be stably recruited to ER microsomes after activation by the nonhydrolyzable analogue of GTP, GTPγS. Whereas wild-type Sar1 was efficiently recruited to the ER after activation with GTPγS ( A, inset, compare a with b ) as observed previously (Aridor et al., 1995
), in the presence of GTPγS, the Sar1[AAAA] mutant failed to be recruited over levels observed in the GDP control ( A, inset, compare c with d). Thus, stable membrane recruitment to the ER membrane is sensitive to mutation of the α1′ helix.
Given the importance of the membrane-associated mammalian mSec12 GEF for Sar1 activation leading to COPII vesicle formation in vitro (Weissman et al., 2001
), we examined the effect of the [AAAA] substitution on interaction of Sar1 with purified mSec12 GEF in the presence of detergent. Exchange was measured by preloading Sar1 with [3
H]-GDP and incubating in the presence of 1 mM cold GTP in the absence or presence of mSec12 (Weissman et al., 2001
). mSec12 promoted efficient nucleotide exchange on wild-type Sar1 ( B). Moreover, the Sar1[AAAA] mutant had a comparable rate of mSec12-stimulated exchange. Because the mutation of the α1′ helix did not prevent interaction with mSec12, the inability of Sar1 to be activated by ER-associated mSec12 is not the explanation for its inability to support COPII vesicle budding in vitro.
The next step in COPII coat assembly from mammalian ER microsomes following activation of Sar1 is the recruitment of the cytosolic Sec23/24 coat complex to form detergent-resistant, prebudding complexes (Aridor et al., 1998
). Therefore, we examined whether the α1′ helix was required for interaction with Sec23/24. As the Sec23/24 complex is a Sar1-specific GAP (Hicke and Schekman, 1989
; Yoshihisa et al., 1993
; Aridor et al., 1998
), direct interaction between these proteins can be determined by examining the ability of purified mammalian Sec23/24 to accelerate GTP hydrolysis of activated Sar1 (Aridor et al., 1998
). Wild-type Sar1 or the Sar1[AAAA] mutant were incubated the presence of purified Sec23/24 GAP and α[32
P]GTP, and the amount of radiolabeled GDP formed determined (Aridor et al., 1998
). Whereas Sec23/24 promoted rapid GTP hydrolysis of wild-type Sar1, the Sar1[AAAA] mutant showed a markedly reduced rate and extent of Sec23/24 promoted GTP hydrolysis ( C). These results suggest that the failure of the Sar1[AAAA] mutant to be stably recruited to ER membranes ( A) to form COPII prebudding complexes may be related to its inability to interact with Sec23/24. This interpretation is consistent with previous results in which stable recruitment of activated Sar1 to ER membranes requires Sec23/24, which is essential to form detergent resistant prebudding complexes (Aridor et al., 1998
). Thus, the α1′ helix functions to facilitate coat assembly on ER membranes.
An NH2-terminal extension appended to the α1′ helix of Sar1 is required for function in vitro and in vivo
The COOH-terminal extensions found in Ras, Rho/Rac/Cdc42, and Rab-like GTPases contain prenyl-lipid modifications promoting membrane association (Seabra, 1998
). In contrast, Arf family members contain a myristoyl modification attached to the Gly residue found at the NH2
terminus of the α1′ helix (Kahn et al., 1988
A). These lipid modifications are essential for both membrane association and function.
Figure 3. Sar1 NH2 terminus is required for function in vitro. (A) Alignment of NH2-terminal regions of representative Ras superfamily GTPases showing the unique NH2-terminal extension found in Sar1. The α1′ helix indicated is that of Sar1. The (more ...)
Curiously, Sar1 lacks any known lipid modifications at either termini (unpublished data). However, appended to the NH2
terminus of Sar1 is a unique extension not found in Arf family or other Ras superfamily members ( A). By analogy to the proposed role of the myristoyl group in Arf1 for bilayer association (Antonny et al., 1997
; Beraud-Dufour et al., 1999
), one possibility is that the NH2
-terminal extension of Sar1 contains information necessary for its binding to ER membranes to initiate COPII vesicle formation. Indeed, whereas wild-type Sar1 or the GTPase-defective mutant Sar1[H79G] support efficient COPII vesicle formation in vitro, a deletion mutant lacking residues 2–9 (Δ9-Sar1) or a Δ9-Sar1[H79G] double mutant (a mutant containing both the Δ9 truncation and the H79G substitution), were unable to support COPII vesicle budding in vitro ( B). Moreover, whereas ER vesicle formation in the presence of crude cytosol (which contains all three COPII components) can be blocked with the dominant negative GDP-restricted form of Sar1, Sar1[T39N] (Kuge et al., 1994
; Aridor et al., 1995
; Weissman et al., 2001
), the double mutant, Δ9-Sar1[T39N] (a mutant containing both the Δ9 truncation and the T39N substitution), did not block vesicle formation ( B). A similar result was observed in vivo where the ability of the transiently expressed Sar1[T39N] to block ER to Golgi transport was lost in the Δ9-Sar1[T39N] double mutant (unpublished data). Because Sar1[T39N] is an inhibitor of the Sar1-specific GEF (Weissman et al., 2001
), we conclude that the NH2
-terminal extension participates in the initial events of COPII vesicle budding related to Sar1 recruitment and/or activation.
A hydrophobic patch within the NH2 terminus is critical for ER export
Given the importance of the NH2-terminal region in mammalian Sar1 function ( B), we examined whether the extension was found in evolutionarily divergent Sar1 GTPases. Remarkably, sequence alignment of divergent members of the Sar1 family illustrated that they not only contained an NH2-terminal extension, but the alignment revealed a highly conserved bulky hydrophobic patch (
A). Therefore, to accurately define the region(s) of importance in the Sar1 NH2 terminus in function, we characterized the activities of more limited NH2-terminal truncations on VSV-G export into COPII vesicles in vitro ( B). Whereas deletion of amino acids 1–3 or 1–4 (Δ3, Δ4-Sar1) only weakly interfered with vesicle budding, strikingly, deletion beyond residues 5 (Δ5-Sar1) potently blocked vesicle formation. Thus, the hydrophobic patch appears to contribute significantly to COPII vesicle budding.
Figure 4. The NH2 terminus of Sar1 is evolutionarily conserved and essential for function. (A) Sequence alignment of the NH2-terminal residues of Sar1 GTPases (C.g., Cricetulus griseus [Chinese hamster]; C.e., Caenorhabditis elegans; D.m., Drosophila (more ...)
To determine whether the patch was also required for function in lower eukaryotes, we made deletions in the Saccharomyces cervisiae homologue of Sar1 (scSar1p) based on the sequence alignment of Sar1 homologs ( A). We focused on only two deletion mutants: Δ9-scSar1p corresponding to the mammalian Δ9-Sar1 truncation that lacked activity ( B), and Δ6-scSar1p corresponding to the mammalian Δ4-Sar1 mutant that retained significant activity ( B). When we introduced these two yeast constructs into a strain that lacks the Sar1 gene (ΔSar1), complementation was observed with Δ6-scSar1p, but not with the Δ9-scSar1p ( C). Thus, in agreement with the important role of the hydrophobic patch in ER export in vivo and in vitro in mammalian cells, the short sequence of bulky hydrophobic residues defines an evolutionarily conserved function.
The hydrophobic patch serves a motif for Sar1 function
The above experiments suggest that either the overall length of the NH2
terminus is critical for Sar1 activity, or that the short, evolutionarily conserved patch encompassing several bulky hydrophobic residues is important for function (). To disrupt function of this patch, we chose a hydrophilic Asp residue to replace Phe 5 as deletion of 4 residues had little effect on Sar1 function, whereas deletion of 5 residues led to striking loss of function ( B). Strikingly, when we tested the ability of the Sar1[F5D] mutant to support budding in vitro, the mutant was unable to reconstitute COPII vesicle formation (
A). Moreover, it did not interfere with vesicle formation in the presence of cytosol-containing wild-type Sar1, demonstrating that the [F5D] mutant was not dominant negative ( A) like the [T39N] or [H79G] mutants. In addition, while Sar1[T39N] showed dominant negative activity in preventing vesicle budding in vitro ( A), the Sar1[T39N][F5D] double mutant lost its ability to function as a dominant negative inhibitor in vitro ( A) and in vivo (unpublished data). This result raises the possibility that the [F5D] substitution may uncouple the ability of the Sar1[T39N] mutant to interact with its specific ER-localized exchange factor mSec12 (Weissman et al., 2001
Figure 5. The hydrophobic patch at the NH2 terminus of Sar1 is essential for recognition of Sec23/24. (A) ER microsomes were incubated in the presence of ATP, GTP, and rat liver cytosol or purified COPII components for 30 min at 32°C, and then the amount (more ...)
In contrast to the [F5D] substitution, mutation of Phe 5 to Ala ([F5A]) had no effect on the wild-type activity of Sar1 to support vesicle budding in vitro ( A), or on the ability of the Sar1[T39N] mutant to inhibit ER to Golgi transport in vitro ( A) and in vivo (unpublished data). Thus, when mutated to Ala, loss of Phe5 function can be compensated by adjacent bulky hydrophobic residues suggesting that Phe3 and Ile4 may contribute to the formation of a general hydrophobic patch that promotes association with the lipid bilayer. Consistent with this possibly, a triple AAA substitution for residues Phe 3, Ile 4, and Phe 5, that would remove all of the bulky hydrophobic residues, had an equivalent phenotype to the Sar1[F5D] mutant ( A). Surprisingly, though, when Phe 3 was mutated to Asp (Sar1[F3D]) leaving the bulky Ile4 and Phe5 residues, Sar1 supported efficient COPII vesicle budding ( A). Thus, Phe3 and Phe5 differ in function. Given the symmetry of the hydrophobic residues (FIF), this would not be expected if the two Phe residues simply functioned similar to a lipid group (such as the myristoyl group found in Arf) for promoting nonspecific bilayer association. These results favor the interpretation that the hydrophobic patch contains sequence specific information that is necessary for Sar1 activity in COP II mediated budding from ER microsomes.
The hydrophobic motif is not essential for nucleotide association or Sec23/24 promoted GTP-hydrolysis
The Phe5 to Asp substitution in the hydrophobic motif could lead to a loss of guanine nucleotide binding, a defect in interaction with the membrane or membrane-associated receptors (such as the membrane-associated mSec12 GEF), or interference with interaction with the cytosolic Sec23/24 complex. To further explore the function of the hydrophobic motif, we first determined that loss of activity was not due to misfolding of the core-fold of Sar1 because the F5D substitution had no effect on nucleotide binding (Fig. S2 B, available at http://www.jcb.org/cgi/content/full/200106039/DC1
). To determine if Sar1[F5D] was responsive to the Sar1-specific Sec23/24 GAP complex, we incubated wild-type Sar1 or the Sar1[F5D] mutant with purified Sec23/24 in the presence of α[32
P]GTP. In both cases, the Sec23/24 complex stimulated the rate of Sar1 GTP hydrolysis to levels previously observed for wild-type Sar1 ( B) (Aridor et al., 1998
). Thus, neither intrinsic GDP/GTP binding nor Sec23/24 stimulated GTP hydrolysis, as can be measured by the above assays, are responsible for the defect in function of Sar1[F5D]. The latter results differ strikingly from the effect of mutation of the α1′ helix to the Sar1[AAAA] mutant form ( C), suggesting that the NH2
terminus contains at least two regions that dictate Sar1 function.
The hydrophobic motif is required for stable recruitment of Sar1 to ER membranes
To determine if the hydrophobic motif was involved in Sar1 recruitment to ER membranes, we incubated purified wild-type Sar1 and the Sar1[F5D] mutant with ER microsomes in the presence of GDP, GTP, or GTPγS. Membranes were then pelleted by centrifugation, and membrane and soluble fractions were separated by SDS-PAGE, immunoblotted, and quantitated for Sar1. Very little binding of wild-type Sar1 or the F5D mutant to ER membranes occurs on ice (Aridor et al., 1995
; Aridor and Balch, 2000
A), indicating the need for physiological activation of Sar1 by the ER-localized Sar1-specific mSec12 GEF. Upon incubation at 32°C, >50% of total wild-type Sar1 added to the assay was stably recruited to membranes in the presence of GTPγS ( A). In contrast, recruitment was not observed with the Sar1[F5D] mutant as <5–10% of Sar1[F5D] was bound under the same conditions ( A).
Figure 6. Sar1[F5D] is defective in stable membrane recruitment and recognition of mSec12. (A and B) Microsomes were incubated on ice or at 32°C in the presence of ATP (lanes 1–10) or no ATP (lane 11), and either 1 μg Sar1, 1 μg (more ...)
To determine whether loss of stable membrane association by Sar1[F5D] was not simply due to the absence of Sec23/24 in the binding assay, we examined the ability of Sar1 to recruit Sec23/24 to ER microsomes. While wild-type Sar1 was able to efficiently recruit Sec23/24 in the presence of GTP or GTPγS, as shown previously (Aridor et al., 1995
) ( B), incubation of ER microsomes with the Sar1[F5D] mutant in the presence of Sec3/24 and GTP or GTPγS only weakly recruited Sec23/24 ( B). Given that the Sar1[F5D] mutant is not defective in its interaction with the Sec23/24 ( B), it is apparent that the hydrophobic motif facilitates stable recruitment of both Sar1 and Sec23/24 to ER membranes, and that recruitment of Sar1 through the hydrophobic motif precedes stable recruitment of Sec23/24.
The hydrophobic motif facilitates Sar1 activation by the mSec12 exchange factor
Given the inability of the Sar1[F5D] mutant to stably bind membranes ( A) even though interaction with Sec23/24 was normal ( C), we examined the effect of the [F5D] substitution on activation by purified mSec12 GEF in the presence of detergent (Weissman et al., 2001
). In contrast to the ability of mSec12 to promote efficient exchange on wild-type Sar1, the Sar1[F5D] mutant was significantly reduced (75% or three- to fourfold) in its rate of exchange in Sec12 GEF stimulated nucleotide exchange ( C). Strikingly, the Δ5-Sar1 truncation completely lost its ability to interact with Sec12 ( C, right panel).
Although the sequence specificity of the FIF motif in COPII vesicle formation suggests that it may be a targeting motif ( A), it remained possible that the FIF sequence could simply promote partitioning of Sar1 into a detergent micelle containing mSec12 in the exchange assay, thereby facilitating efficient exchange. To address this concern, we generated a detergent-free preparation of mSec12 and analyzed the ability of mSec12 to promote exchange in the absence of lipid or detergent. In a detergent-free exchange reaction, we observed no loss of activity of mSec12 in promoting efficient exchange from wild-type Sar1 ( C). Importantly, the inability of the [F5D] mutant to support exchange was identical to the level observed in the presence of detergent ( C). The importance of the NH2-terminal region for Sar1 activation by Sec12 was further demonstrated by generation of Δ25-Sar1, a truncation mutant lacking the entire NH2
terminus, including the α1′ helix (equivalent to Arf1 [Δ17]). As expected, this truncation has lost its ability to interact with Sec12 (Fig. S4, available at http://www.jcb.org/cgi/content/full/200106039/DC1
). Thus, Sar1 requires a conserved hydrophobic motif found in the NH2
terminus to provide a domain that facilitates activation by the ER-associated mSec12. Given these results, we now refer to this short cluster of bulky hydrophobic amino acids as the STAR motif reflecting its role as a Sar1–NH2
-terminal activation recruitment sequence.