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Vol. 14, Issue 8, 3114-3125, August 2003
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Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel
Submitted December 10, 2002;
Revised March 16, 2003;
Accepted April 2, 2003
Monitoring Editor: Keith Mostov
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). Cognate SNAREs present on both donor and acceptor membranes
(designated as v-SNAREs and t-SNAREs, respectively), interact in
trans to form a tight four-helix bundle
(Sutton et al.,
1998), which brings the bilayers into proximity and is thought to
lead to membrane fusion (Lin and Scheller,
2000; Waters and Hughson;
2000). After fusion, SNAREs reside in the same membrane in an
inactive cis conformation. Soluble
N-ethylmaleimide-sensitive fusion protein (NSF) acts to disassemble
cis complexes and primes the SNAREs for the next round of fusion
(Lin and Scheller, 2000;
Wickner and Haas, 2000).
Although other factors may be involved in vivo
(Peters et al.,
2001), SNAREs constitute the minimal machinery required for
membrane fusion in vitro (Weber et
al., 1998).
The events leading to SNARE assembly in vivo involve a number of accessory
proteins, though their functions are not entirely known. For example,
Sec1/Munc18 (SM) interacts with members of the Sso/syntaxin family of t-SNAREs
(Hata et al., 1993;
Garcia et al., 1994;
Pevsner et al.,
1994). Yeast Sec1 binds to exocytic SNARE complexes containing
Sso, Sec9 (a SNAP-25 ortholog), and Snc (a VAMP ortholog), but apparently not
to free Sso (Carr et al.,
1999). Sly1, a Sec1 homolog that acts upon endoplasmic reticulum
(ER)-Golgi transport, binds to the Sed5 t-SNARE, and confers specificity to
SNARE assembly (Peng and Gallwitz,
2002). Likewise, Vps45 (an endosomal Sec1 homolog) associates with
the Tlg2 endocytic t-SNARE and facilitates assembly with Tlg1 and Vti1
(Bryant and James, 2001). Thus,
yeast SM proteins act in a positive manner to confer complex formation.
Although mammalian Sec1 binds to free and uncomplexed syntaxin in vitro
(Pevsner et al.,
1994; Yang et al.,
2000) and maintains the t-SNARE in a "closed" inactive
conformation (Nicholson et al.,
1998), recent studies in animal cells also suggest that SM
proteins facilitate assembly (Verhage
et al., 2000). Another SNARE regulator, LMA1, binds to
the primed vacuolar t-SNARE, Vam3, to prevent the reformation of cis
SNARE complexes (Xu et al.,
1998; Wickner and Haas,
2000). The release of LMA1 from Vam3 is, therefore, necessary to
fusion to proceed. Our laboratory identified Vsm1/Ddi1 as a negative regulator
of the exocytic SNAREs in yeast
(Lustgarten and Gerst, 1999).
Vsm1 binds directly to the Snc1,2 v-SNAREs and its overproduction results in
the accumulation of low-density secretory vesicles, coupled with an inhibition
of growth and secretion in cells bearing a mutant Sec9 t-SNARE.
Both SNAREs and SNARE regulatory proteins are phosphorylated in vitro (for
reviews, see Gerst, 1999;
Lin and Scheller, 2000). For
instance, the phosphorylation of syntaxins by protein kinase C, protein kinase
A (PKA), and other kinases reduces their affinity for SNARE partners, while
increasing their affinity for the synaptotagmin SNARE regulator
(Gerst, 1999;
Lin and Scheller, 2000).
Studies on vacuolar fusion in yeast have shown that the release of LMA1 from
Vam3 is dependent upon the function of protein phosphatase 1 (PP1)
(Peters et al.,
1999). Thus, the dephosphorylation of Vam3 may be necessary for
vacuolar fusion (Wickner and Haas,
2000). Recently, we demonstrated that phosphorylation of the Sso
exocytic t-SNAREs by PKA inhibits SNARE assembly and vesicle fusion
(Marash and Gerst, 2001).
Mutation of a PKA site (serine-79) to alanine in the autoinhibitory domain of
Sso1, or its dephosphorylation by a ceramide-activated protein phosphatase
(CAPP), increased the ability of Sso to assemble into complexes with the Sec9
t-SNARE. This restored exocytosis and normal growth in certain secretion
mutants. Similarly, phosphorylation of the endosomal Tlg t-SNAREs was found to
inhibit Tlg SNARE assembly and endocytosis
(Gurunathan et al.,
2002). Thus, phosphorylation plays a critical role in regulating
the availability of t-SNAREs to assemble into v-t SNARE complexes and to
confer membrane fusion in vivo.
We now report that the mechanism of phosphorylation-dependent inhibition of the exocytic t-SNAREs in yeast may involve the recruitment of a SNARE regulator. Phosphorylation of the Sso t-SNAREs by PKA increases their affinity for Vsm1 and modulates its binding both in vivo and in vitro. Moreover, Vsm1 binding to this t-SNARE may reduce the availability of Sso to bind the Sec9 t-SNARE and, thus, inhibit SNARE assembly. In contrast, unphosphorylated Sso shows a reduced ability to bind Vsm1 and an increased ability to interact with Sec9, both in vivo and in vitro. Thus, Vsm1 binding may be mutually exclusive with Sec9 binding. Vsm1 requires the NH2-terminal autoinhibitory domain of the t-SNARE for this interaction and constitutively "open" conformations of Sso1 show a reduced ability to bind Vsm1. Finally, removal of the ubiquitin-association domain (UBA) of Vsm1 did not affect Sso binding, and neither VSM1 overexpression nor deletion altered t-SNARE stability. Thus, Vsm1 does not regulate Sso degradation, but it may modulate the ability of the t-SNARE to enter into functional SNARE complexes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Plasmids
Plasmids used in this work are listed in
Table 2.
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Protein Phosphorylation
Glutathione S-transferase (GST)-Sso11-265,
GST-Sso11-265,A66, GST-Sso11-265,A79, and
His6-Vsm1 (3.4E-11 moles each) were phosphorylated with (up to 4
µg) Tpk1 in the presence of 50 µCi of [
-32P]ATP (5
Ci/µmol) and visualized as described previously
(Marash and Gerst, 2001).
Pulse-Chase Analysis
Intracellular processing of Sso was monitored by pulse-chase analysis by
using [35S]methionine (Amersham Biosciences, Piscataway, NJ), as
described previously (Couve et
al., 1995).
Measurement of Protein Complexes
Immunoprecipitation (IP) from Lysates. IP of both SNARE and
Sso-Vsm1 complexes from total cell lysates (TCLs) was performed by
coimmunoprecipitation, by using the modifications described in
Marash and Gerst, 2001.
Anti-myc (Santa Cruz Biotechnology, Santa Cruz, CA) and anti-Vsm1
(Lustgarten and Gerst, 1999)
antibodies (abs) were used for IP (4 and 1 µl/reaction, respectively).
Samples of TCLs and IPs were resolved by SDS-PAGE and detected by Western
analysis to determine the amount of Sec9, Snc, Sso, and Vsm1 that either
immunoprecipitated or coimmunoprecipitated with a given antiserum. Antibodies
used for detection included anti-phosphoserine (1:1000) (Zymed Laboratories,
South San Francisco, CA); anti-Sso (1:3000) (gift of S. Keranen, VTT
Biotechnology, Espoo, Finland), anti-Sec9 (1:1000) (C terminus) (gift of P.
Brennwald, University of North Carolina, Chapel Hill, NC), anti-Snc (1:500)
(Protopopov et al.,
1993), anti-Sed5 (1:3000) and anti-Tlg1 (1:2000) (gifts of D.
Banfield, Hong Kong University of Science and Technology, Hong Kong, China),
anti-Tlg2 (1:2000) (gift of H. Abeliovich, Hebrew University of Jerusalem,
Rehovot, Israel), anti-Vsm1 (1:3000)
(Lustgarten and Gerst, 1999),
and anti-Vti1 antibodies (1:3000) (gift of G. Fischer von Mollard,
Georg-August Universitat Gottingen, Germany). Detection was performed by
chemiluminescence. To improve detection and accuracy, TCL samples were
normalized to account for variations in protein expression and recovery.
Sso-Vsm1 Assembly In Vitro. Recombinant affinity-purified GST-Sso11-265, GST-Sso11-265,A66, and GST-Sso11-265,A79 (phosphorylated or nonphosphorylated) and His6-Vsm1 proteins were mixed together at a ratio 1:1 (3.4E-11 moles) in buffer containing 0.5% NP-40 in phosphate-buffered saline, and allowed to incubate overnight at 4°C. Thereafter, proteins were immunoprecipitated with anti-Vsm1 abs (1 µl/reaction), resolved by SDS-PAGE, and detected in blots with anti-Vsm1 and anti-Sso abs (1:3000).
Sso-Sec9 Assembly In Vitro. Purified GST-Sso11-265 (2E-11 moles) and GST-Sec9402-651 (1E-11 moles) were incubated in the absence or presence of increasing amounts of His6-Vsm1 (0.2–10E-11 moles) at 4°C, and resolved by IP and SDS-PAGE (Figure 1C). For competition binding studies (Figure 5), GST-Sso11-265 and GST-Sso11-265,A79 were mixed together at different ratios (0:1, 0.25:0.75, 0.5:0.5, 0.75: 0.25, and 1:0) to yield a final concentration of 3E-11 moles and incubated with 3E-11 moles each of His6-Vsm1 and GST-Sec9402-651.
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For both experiments, complexes were immunoprecipitated using anti-Sso abs (1 µl/reaction) and detected quantitatively in Westerns by using anti-Sec9 (1:1000), -Vsm1 (1:3000), or -Sso (1:3000) abs.
Measurement of Sso1-Vsm1 Stoichiometry In Vitro. Moles (2.6E-11) of either GST-Sso11-265 or GST-Sso11-265,D79 were mixed with increasing concentrations of His6-Vsm1 (between 1 and 16E-11 moles) and incubated overnight at 4°C in buffer containing 0.5% NP-40 in phosphate-buffered saline. Proteins were then subjected to IP with anti-Sso abs (1 µl/reaction), resolved by SDS-PAGE, and detected quantitatively in blots by using anti-Sso and anti-Vsm1 (1:3000) abs. Molar quantification of the proteins was determined using known quantities of GST-Sso11-265 and His6-Vsm1 that were purified over glutathione-Sepharose, or nickel beads (Pharmacia, Peapack, NJ), and electrophoresed and detected in parallel to the immunoprecipitated proteins.
Measurement of Sso1-Vsm1 Affinity In Vitro. To measure the affinity of the Sso1–Vsm1 interaction, 0.2E-11 moles of His6-Vsm1 and 0.8E-11 moles of either GST-Sso11-265 or GST-Sso11-265,D79 was incubated with increasing amounts of His6-HA-Vsm1 (e.g., 0, 0.3-, 1.3-, 3.1-, 8.7-, 15.8-, and 31.8E-11 moles). Complexes were precipitated using glutathione-Sepharose beads, separated by SDS-PAGE, and detected with either anti-Sso or anti-Vsm1 abs (1:3000) in blots. Molar quantification of the proteins was determined using known amounts of purified GST-Sso11-265, His6-Vsm1, and His6-HAVsm1 that were detected in parallel to the immunoprecipitated proteins. The constant for half-maximal binding of Vsm1 to Sso was calculated by measuring the displacement of His6-Vsm1 by His6-HAVsm1 in the Sso1-containing complexes. Inverse reciprocal plots of the molar amounts of bound Vsm1 (His6-HAVsm1 and His6-Vsm1) (y-axis) versus total added Vsm1 (x-axis) were made and the data subjected to linear regression. The absolute value of the crossing point of the regression line with the x-axis represents the inverse value of the binding constant.
Measurement of the Rate of Sso1-Vsm1 Binding In Vitro. Equal amounts of His6-Vsm1 (1.5E-11 moles) were added to either GST-Sso11-265 or GST-Sso11-265,D79 (3.4E-11 moles) and incubated for 0, 2, 4, 8, or 16 h. Protein complexes were precipitated with glutathione-Sepharose beads, resolved by SDS-PAGE, and detected with either anti-Sso or anti-Vsm1 abs (1:3000) in blots. Molar quantification of the proteins was determined using known amounts of GST-Sso11-265, His6-Vsm1, and GST-Sso11-265,D79 that were detected in parallel to the immunoprecipitated proteins.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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).
Deletion of VSM1 in wild-type (WT) cells increases the secretion of
proteins into medium, whereas its overexpression leads to the repression of
cell growth and secretion, particularly in sec9-4 cells. In addition,
direct physical interactions between Vsm1 and the Snc v-SNAREs were observed,
suggesting that Vsm1 might exert its effect upon the secretory pathway via the
regulation of v-SNARE function. In studies designed to show the specificity of Vsm1 binding to the Snc v-SNAREs, we examined whether it also binds the Sso t-SNAREs. We precipitated Vsm1 from lysates prepared from WT cells expressing myc-tagged Sso1 and looked for the presence of the t-SNARE. Surprisingly, we found that Sso coimmunoprecipitates with Vsm1 (Figure 1B), suggesting that these t-SNAREs may be targets for Vsm1.
To help define the region of Sso required for Vsm1 binding, we constructed
deletion mutants and expressed them in yeast
(Figure 1, A and B). We tested
myc-tagged mutants bearing either a partial (
1;
Sso11-103) or full (2;
Sso11-146) deletion of the
NH2-terminal autoinhibitory domain, as well as one lacking the
COOH-terminal SNARE binding domain (Sso12-46). Next, we
immunoprecipitated complexes formed between the Sso1 deletion mutants and
Vsm1, by using anti-Vsm1 antibodies. We found that partial deletion of the
NH2-terminal of Sso1 greatly reduced (by >20-fold) the binding
of Sso1 to Vsm1, whereas deletion of the entire autoinhibitory domain resulted
in complete abrogation of the interaction
(Figure 1B). This suggests that
the NH2-terminal domain of Sso1 is necessary for Vsm1 binding.
The autoinhibitory domain of Sso interacts with the SNARE-binding motif of
the t-SNARE to form an intramolecular closed structure, thus, preventing SNARE
complex assembly (Nicholson et
al., 1998). Because this domain may be necessary for Vsm1
binding, we examined whether Vsm1 binds directly to it when expressed alone in
yeast (Figure 1B). To ensure
proper localization of the NH2-terminal fragment
(Sso12-46) to the plasma membrane, we inserted a CAAX motif at its
COOH terminus. Expression of this construct in WT cells had no deleterious
effects upon growth (our unpublished data). Upon immunoprecipitation with
anti-Vsm1 antibodies, we found that a portion of the autoinhibitory domain was
bound to Vsm1. This suggests that the SNARE-binding domain of Sso is not
required for its association with the Vsm1 SNARE regulator.
To determine whether Vsm1 binding affects the ability of Sso to bind to its t-SNARE partner, Sec9, we examined whether increasing amounts of Vsm1 could displace Sec9 binding in vitro. We used increasing amounts of recombinant His6-tagged Vsm1 (0.2–10E-10 moles) in binding reactions containing GST-tagged Sso11-265 and GST-Sec9402-651 (2E-11 and 1E-11 moles, respectively). As the concentration of Vsm1 in the assay increased, there was a corresponding decrease in the amount of Sec9 bound to Sso1 (Figure 1C). Thus, Vsm1 acts as a competitive inhibitor of SNARE assembly in vitro.
To determine stoichiometry of the Sso-Vsm1 interaction, we incubated fixed amounts of recombinant GST-Sso11-265 (2.3E-11 moles) with increasing amounts of His6-Vsm1 (0, 1, 2, 4, 8, and 16E-11 moles). To measure the molar equivalents of precipitated His6-Vsm1 and bound GST-Sso11-265, we used purified GST-Sso11-265 and His6-Vsm1 as protein standards that were detected in parallel in Westerns. This quantitative analysis revealed that His6-Vsm1 and GST-Sso11-265 interact in vitro and at a ratio of 1:1, at saturation (Figure 1D).
Phosphorylation of Sso1 at Serine-79 by Tpk1 Promotes Vsm1 Binding In
Vitro
Given that PKA phosphorylation of the NH2 terminus domain of the
Sso t-SNAREs inhibits SNARE assembly and that Vsm1 binds to this domain, we
determined the effect of phosphorylation upon Vsm1 binding in vitro
(Figure 2A). Purified
GST-Sso11-265 (3.4E-11 moles) was phosphorylated in vitro with
unlabeled ATP by using increasing amounts of recombinant Tpk1 (a catalytic
subunit of PKA) and mixed with an equimolar amount of His6-Vsm1.
Next, complexes were precipitated with anti-Sso antibodies and the amount of
bound Vsm1 measured by quantitative Western analysis. In parallel, equal
amounts of GST-Sso11-265 and His6-Vsm1 were
phosphorylated under identical conditions by using radioactive
[-32P]ATP. Interestingly, we found that an increase in Sso
phosphorylation correlated with a large enhancement in Vsm1 binding
(Figure 2A), whereas Vsm1
itself was not phosphorylated under these conditions
(Figure 2A) or in vivo
(Lustgarten and Gerst,
1999).
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Analysis of the Sso1 sequence reveals two putative PKA phosphorylation
sites at threonine-66 and serine-79, although only serine-79 plays a role in
SNARE assembly (Marash and Gerst,
2001). To define the extent of GST-Sso11-265
phosphorylation, we found that 1.9 moles of phosphate was incorporated per
mole of GST-Sso11-265 (7.6E-11 moles of phosphate was incorporated
into 3.96E-11 moles of Sso1) at saturation, suggesting that both PKA sites
undergo phosphorylation by Tpk1 (our unpublished data;
Marash and Gerst, 2001). To
determine which PKA site plays a role in the binding of Vsm1, we
phosphorylated mutants bearing alanine substitutions at the relevant PKA sites
of Sso1 (e.g., GST-Sso11-265,A66 and GST-Sso11-265,A79)
in vitro. The phosphorylated t-SNAREs were then mixed with
His6-Vsm1 and immunoprecipitated with anti-Vsm1 antibodies. As
shown above, Tpk1-dependent phosphorylation of GST-Sso11-265
increased the binding of Vsm1 (Figure 2, A
and B). Presence of the alanine substitution at position 66 of
Sso1 did not inhibit the interaction
(Figure 2B), whereas, in
contrast, an alanine substitution at position 79 decreased Vsm1 binding by
threefold, even in the presence of Tpk1. Alanine substitutions at both
positions did not inhibit the Vsm1–Sso1 interaction more than the single
substitution at position 79 (our unpublished data). Thus, the phosphorylation
of Sso1 at serine-79 is likely to be important for normal Vsm1 binding.
As shown above (Figure 1D), unphosphorylated Sso binds Vsm1 at a 1:1 ratio. To determine whether phosphorylation of serine-79 alters the stoichiometry of the Sso–Vsm1 interaction, we introduced an aspartate mutation that mimics phosphorylation into this site. We incubated fixed amounts of purified recombinant GST-Sso11-265,D79 (2.3E-11 moles) with increasing amounts of His6-Vsm1 (0, 1, 2, 4, 8, and 16E-11 moles) (Figure 2C). After precipitation and quantitative detection in blots, we found that His6-Vsm1 and GST-Sso11-265,D79 also interact in vitro in a ratio of 1:1.
Because the stoichiometries of Vsm1 binding to native Sso1 or Sso1D79 are equivalent, we examined whether the increased binding of Vsm1 to phosphorylated Sso1 results from a change in affinity. To measure the binding affinities of Sso1 and pseudophosphorylated Sso1 for Vsm1, we incubated a mixture of 0.2E-11 moles of His6-Vsm1 and 0.8E-11 moles of either GST-Sso11-265or GST-Sso11-265,D79 with increasing amounts of His6-HAVsm1 (e.g., 0–31.8E-11 moles) (Figure 3A). After quantitative detection, the constants for half-maximal binding were calculated from the molar concentrations of His6-HAVsm1 necessary to displace His6-Vsm1 in Sso1- and Sso1D79-containing complexes. We found that the affinity of Vsm1 for native Sso1 is fivefold lower (n = 2 experiments) than for Sso1D79 (1.9E-7M versus 4.2E-8M in the experiment shown in Figure 3A). This suggests that Sso phosphorylation greatly increases its affinity for Vsm1.
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To further characterize the effect of Sso phosphorylation on Vsm1 binding, we measured the rate of assembly between Vsm1 and either Sso11-265 or Sso11-265,D79. We added 1.5E-11 moles of His6-Vsm1 to equal amounts (3.4E-11 moles) of either GST-Sso11-265 or GST-Sso11-265,D79 and incubated them for 0–16 h (Figure 3B). We found that Vsm1 binding to Sso11-265,D79 reached saturation in half the time it took with native Sso1. Thus, Sso phosphorylation increases both the affinity for, and rate of assembly with, Vsm1.
Phosphorylation of Sso1 at Serine-79 Promotes Vsm1 Binding In
Vivo
PKA-dependent phosphorylation of Sso inhibits SNARE complex formation by
reducing the apparent affinity for its t-SNARE partner, Sec9
(Marash and Gerst, 2001). This
work suggests that the phosphorylation of Sso1 on serine-79 has an additional
effect, i.e., enhancing the binding of Vsm1 to Sso1 in vitro. To find out
whether this effect is meaningful in living cells, we examined Vsm1 binding to
Sso1 and the Sso1 alanine substitution mutants in vivo. We expressed
myc-tagged forms of native Sso1, Sso1A66, and Sso1A79 in
WT cells and examined their ability to bind Vsm1. We found that both native
and mutant Sso1 bound Vsm1 (Figure
4A); however, Sso1A79 bound about half as much as
either native Sso1 or Sso1A66. Because the alanine-79 mutant is
significantly less phosphorylated in vivo
(Marash and Gerst, 2001), it
suggested that phosphorylation mediates the Vsm1-Sso1 interaction in vivo, as
well as in vitro (Figures 2, A and
B, and 3). To
determine whether Sso bound to Vsm1 is phosphorylated, we used an
anti-phosphoserine antibody that can be used to detect SNAREs that undergo
phosphorylation in vivo (our unpublished data). When precipitated using
anti-Vsm1 antibodies we found that native Sso1, as well as the
Sso1A66 and Sso1A79 mutants, could be detected with the
anti-phosphosphserine antibody though the signal corresponding to
Sso1A79 was less. This was expected because the serine-to-alanine
substitution at position 79 blocks the phosphorylation site used by PKA. That
the signal is not completely blocked indicates that other serine residues in
Sso1 may be phosphorylated by additional kinases in vivo. Nonetheless, the
results imply that phosphorylated Sso associates with Vsm1 in vivo.
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Because Sso phosphorylation enhances Vsm1 binding, we conjectured that
t-SNARE dephosphorylation must reduce Sso-Vsm1 assembly. Previously, we showed
that the activation of CAPP dephosphorylates Sso1 at the serine-79 position
(Marash and Gerst, 2001). To
determine whether CAPP activation affects the interaction of native Sso1 and
Sso1A79 with Vsm1, we isolated Sso–Vsm1 complexes from
C2-ceramide–treated and untreated sec9-4 cells
overexpressing SSO1 or SSO1A79. We used
sec9-4 cells because of their enhanced sensitivity to VSM1
overexpression (Lustgarten and Gerst,
1999). Addition of 10 µM C2-ceramide strongly
reduced the binding of Vsm1 to Sso1 (by 3.7-fold), whereas little effect on
the binding to Sso1A79 was seen
(Figure 4B). This result
suggests that CAPP-mediated dephosphorylation of Sso1 at position 79 regulates
its interaction with Vsm1 in vivo.
Vsm1 Does Not Seem to Bind to Sso in SNARE Complexes Formed In
Vivo
Because t-SNARE phosphorylation inhibits SNARE assembly
(Marash and Gerst, 2001), but
mediates the association of Vsm1 and Sso (Figures
2 and
3), it suggested that Vsm1
should not bind to assembled SNARE complexes. This idea is supported by in
vitro binding data showing that Sec9 can be displaced by Vsm1
(Figure 1C). To test this idea
in vivo, we examined Vsm1 binding to Sso in temperature-sensitive
sec18-1 cells, which accumulate SNARE complexes at restrictive
temperatures (Carr et al.,
1999). We found that Vsm1, but not Snc, was bound to Sso at
permissive temperatures (26°C) (Figure
4C). In contrast, a large increase in Snc v-SNARE binding was
observed at 37°C (due to SNARE assembly), whereas no significant change in
the amount of bound Vsm1 was detected, after normalization for the increase in
Sso synthesis and precipitation at 37°C
(Figure 4C). This suggests that
Vsm1 is not likely to bind to Sso t-SNAREs that have assembled into SNARE
complexes in vivo.
Constitutively Open Forms of Sso Bind Less Vsm1
Sso t-SNAREs exist in either an active (open) or inactive (closed)
conformation, mediated by the NH2 terminus autoinhibitory domain
(Nicholson et al.,
1998). Switching from the closed to open conformation is necessary
for Sso assembly into SNARE complexes with its cognate SNARE partners (i.e.,
the Sec9 t-SNAREs and Snc v-SNAREs). To determine which configuration binds
Vsm1, we assessed the ability of Vsm1 to interact with either native Sso1 or
constitutively open mutants (Munson and
Hughson, 2002). The open mutants of Sso1 are fully functional and
are able to replace the native form in yeast. We expected that if Vsm1 binds
preferentially to the closed conformation of Sso1 then native Sso1 should
recruit more Vsm1 than the open mutants. Native Sso1 and the two open
(Sso1V84E,K95E,Y148A [O1] and
Sso1K95A,K99A,R119A,L123A,Y148A [O2]) mutants were expressed in
sso yeast and complexes containing Vsm1 and Sso1 precipitated
using anti-Vsm1 antibodies. We found that Vsm1 binds twice as much native Sso1
than either of the two open forms (Figure
4D). Thus, the open conformation of Sso seems to inhibit Vsm1
binding.
Phosphorylation Alters the Equilibrium between Sso Binding to Its
t-SNARE Partner, Sec9, or to Vsm1
Phosphorylation of Sso inhibits its interaction with Sec9
(Marash and Gerst, 2001) but
increases that with Vsm1 (Figures
2 and
3). Moreover, Vsm1
preferentially binds to the native (Figure
4D) and uncomplexed form of Sso
(Figure 4C) in vivo, and
displaces Sec9 in vitro (Figure
1C). Thus, phosphorylation-dependent Vsm1 binding may prevent Sso
from assembling into SNARE complexes. To verify this, we examined whether the
binding of phosphorylated Sso to Vsm1 excludes Sec9. We mixed
GST-Sso11-265 and GST-Sso11-265,D79 at different ratios
(e.g., 0:1, 0.25:0.75, 0.5:0.5, 0.75:0.25, and 1:0), with equimolar amounts of
His6-Vsm1 and GST-Sec9402-651. Next, we performed
quantitative detection of the amounts of Vsm1 and Sec9402-651 that
precipitated with Sso. We found that as the percentage of
GST-Sso11-265,D79 increased, the amount of Vsm1 bound increased
proportionally (Figure 5, A and
B). Likewise, as the percentage of GST-Sso11-265,D79
decreased, there was a concomitant rise in Sec9 binding. Thus, Vsm1 binds to
Sso at the expense of its SNARE partner.
To determine whether this competition occurs in vivo, we monitored the amounts of Sso1 in complexes with either Vsm1 or Sec9 in sec9-4 cells overexpressing VSM1. Quantitative analysis revealed that 2.2-fold less Vsm1 was bound to Sso1A79 than to native Sso (Figure 6A). Correspondingly, there was a twofold increase in the amount of Sec9 bound to Sso1A79 versus native Sso1. Thus, the reduction in Vsm1 binding of Sso1A79 may lead to an increase in the amount of Sso available to form complexes with Sec9.
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An Alanine Substitution at Position 79 Enhances the Open Conformation
of Sso
Because sec9-4 cells are sensitive to Vsm1 overproduction
(Lustgarten and Gerst, 1999),
we examined whether Sso1, open Sso1 (Sso1-O1), and their alanine-79 mutants
(Sso1A79 and Sso1A79-O1, respectively) rescue
sec9-4 cells overexpressing VSM1, when expressed from
single-copy plasmids. As shown previously, VSM1 overexpression
inhibited the growth of sec9-4 cells
(Figure 6, B and C). However,
Sso1 and Sso1-O1, as well as their alanine-79 mutants, can suppress
Vsm1-mediated growth inhibition (Figure
6B). Notably, rescue by the Sso1A79-O1 mutant was much
stronger than that of either Sso1-O1 or Sso1A79. This suggests that
a lack of phosphorylation at serine-79 enhances functioning of the open (and
active) form of the t-SNARE.
When overexpressed from multicopy plasmids, we found that Sso1A79 also restores the growth of sec9-4 cells overexpressing VSM1 (Figure 6C). This implies, but does not prove, that the alanine mutation at position 79 allows the t-SNARE to assume a more open conformation. Nevertheless, it would seem that the absence of phosphorylation at this site not only reduces the interaction between Sso and Vsm1 in vitro and in vivo, but also leads to the rescue of yeast sensitive to VSM1 overexpression.
Vsm1 Binds to Other Snc-interacting t-SNAREs
To investigate the specificity of Vsm1 interactions with late-acting
SNAREs, we tested its ability to bind to SNAREs involved in other
intracellular fusion events. We immunoprecipitated Vsm1 from WT cells and
examined the precipitates for the presence of Sso, Sed5 (a cis-Golgi
t-SNARE), Vam3 (a vacuolar t-SNARE), Tlg2 (an endosomal t-SNARE), Tlg1 (an
endosomal and trans-Golgi t-SNARE), Snc, and Vti1 (an endosomal
t-SNARE). We found that Vsm1 interacts with Tlg1 and Tlg2, as well as with the
Snc and Sso proteins (Figure
7). Interestingly, Vsm1 was not found in the complexes with SNAREs
that act upon either ER-Golgi or vacuolar fusion. This suggests that Vsm1 acts
upon SNAREs involved in exo- and endocytosis, and specifically with known Snc
v-SNARE partners, e.g., Tlg1, Tlg2, and Sso. That Vsm1 does not interact with
Vti1, a putative component of the endocytic SNARE complex, suggests that its
interactions are specific to certain endosomal t-SNAREs.
|
The UBA Domain of Vsm1 Is Not Required for the Binding to Sso
Vsm1/Ddi1 interacts directly with ubiquitin through a UBA domain at its
COOH terminus (Bertolaet et al.,
2001) and is involved in the degradation of the Pds1 checkpoint
factor (Clarke et al.,
2001). This suggests that Vsm1 regulates the stability of some
proteins. To determine whether Vsm1 modulates t-SNARE stability, we measured
the rate of Sso degradation in sec9-4 cells bearing either a deletion
of VSM1 or overexpressing it from a multi-copy vector. Sso processing
was identical in all cell types and the t-SNARE was stable for up to 3 h
(Figure 8A). This suggests that
Vsm1 does not exert its effect upon the secretory pathway via the modulation
of Sso degradation.
|
To verify this, we constructed a mutant lacking the UBA domain
(Vsm12-402). This mutant should not bind ubiquitin and is unlikely
to participate in degradative processes
(Clarke et al., 2001).
We overexpressed both native and mutant forms of VSM1 in
sec9-4 cells and tested their growth at semirestrictive temperatures
(Figure 8B). We found that the
Vsm1 deletion mutant repressed the growth of sec9-4 cells better than
native Vsm1, suggesting that UBA domain is not necessary for inhibition.
Finally, we tested the binding of native Vsm1 and Vsm12-402 to the Sso t-SNAREs in sec9-4 cells. We found that both forms of Vsm1 bound equally to Sso, suggesting that the UBA domain does not mediate SNARE interactions (Figure 8C). This lends credence to the idea that any role of Vsm1/Ddi1 in protein degradation is irrelevant to its function on the secretory pathway.
|
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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).
Phosphorylation of Sso1 at serine-79 inhibited the interaction with its SNARE
partners, resulting in an inhibition in exocytosis. This effect was observed
in cells lacking the Snc exocytic v-SNAREs, as well as in cells expressing the
full complement of SNAREs, suggesting that the effect of PKA phosphorylation
on SNARE complex assembly is physiologically relevant. Ongoing work has shown
that t-SNARE phosphorylation is not exclusive to the exocytic process but that
it is a general feature of secretory pathway, such as endocytosis
(Gurunathan et al.,
2002) and ER-Golgi transport (our unpublished data). We have found
that the Tlg t-SNAREs, which operate on the endocytic and endosomal transport
routes, are also phosphorylated by PKA, which inhibits SNARE assembly and
endocytosis (Gurunathan et al.,
2002).
Although a role for phosphorylation in the regulation of SNARE assembly is
obvious, the precise mechanism by which it controls complex formation in vivo
is not fully resolved. Herein, we demonstrate that phosphorylation may control
the ability of the Sso t-SNARE to form complexes with Sec9, by modulating the
interaction between Sso and the Vsm1 SNARE regulator. Sso binds directly to
Vsm1 (Figure 1), a
v-SNARE–binding protein and negative regulator of exocytosis
(Lustgarten and Gerst, 1999).
Phosphorylation of Sso1 on serine-79 by PKA increases its affinity for Vsm1
(Figures 2,
3, and
5), leading to a dramatic
reduction of bound Sec9 (Figures
5 and
6A). This was shown using
either phosphorylated Sso1 or an aspartate substitution at position 79.
Correspondingly, substitution of serine-79 with alanine inhibited Vsm1 binding
to Sso in vivo (Figures 4, A and
B, and 6A). Loss of
Sec9 binding to phosphorylated (or pseudophosphorylated) Sso was concommitant
with an increase in Vsm1 binding (Figures
5 and
6A). This suggests that t-SNARE
phosphorylation inhibits the formation of SNARE complexes not only through a
change in affinity (Marash and Gerst,
2001) but also through the recruitment of accessory factors (this
study). Thus, the function of SNARE regulators, such as Vsm1, may be to
control the availability of SNAREs to enter into SNARE complexes.
Vsm1 binding to Sso requires the NH2 terminus of the t-SNARE, because deletion of this regulatory region blocks the interaction. Yet, the NH2 terminus alone binds less Vsm1 than full-length Sso (Figure 1B), suggesting that presence of the COOH-terminal SNARE binding domain yields a structure that is better recognized by Vsm1. Because Vsm1 seems to bind Sso that is not assembled into SNARE complexes (Figures 4C and 5) and displaces Sec9 (Figures 1C and 5), it is likely that Vsm1 interacts preferably with the closed and inactive conformation of Sso, rather than with the active open conformation. This idea is partially supported by tests using specific open conformation mutants of Sso, which bind less Vsm1 than native Sso (Figure 4D). In fact, the lower level of phosphorylation (and loss of Vsm1 binding) seen with the alanine-79 mutant (Figure 4, A and B) correlated well with the rescue of sec9-4 cells (Figure 6C) and greatly enhanced function of an open form of the Sso t-SNARE (Figure 6B). Thus, it may be that dephosphorylation of serine-79 stabilizes the open conformation of Sso (Figure 6C) and leads to Sec9 binding, whereas phosphorylation favors the closed conformation, and leads to Vsm1 binding. More work will be required to verify these points.
This work suggests that PKA positively regulates the Sso–Vsm1
interaction. This is consistent with previous data showing that t-SNARE
phosphorylation regulates the binding of accessory factors. For example,
casein kinase II phosphorylation of syntaxin-4 decreased its affinity for
SNAP-25, but increased the affinity for synaptotagmin
(Risinger and Bennett, 1999).
Because PKA-dependent phosphorylation of Sso1 greatly increased Vsm1 binding
in vitro (Figures 2, A and C,
3, and
5), we assumed that
dephosphorylation would result in its release in vivo. Indeed, CAPP activation
by the ceramide analog C2-ceramide resulted in the release of Vsm1
from Sso (Figure 4B).
Interestingly, dissociation of the LMA1 SNARE regulator from Vam3 depends upon
the activity of the Glc7 phosphatase (PP1). Inhibition of PP1 by the
phosphatase inhibitor microcystin LR blocks the release of LMA1 from Vam3 and
subsequent vacuolar fusion (Peters et
al., 1999; Wickner and
Haas, 2000). Therefore, a precedent for
phosphorylation/dephosphorylation in the binding and release of SNARE
regulatory factors exists, and to which Vsm1 adheres.
Because Vsm1 seems to bind to uncomplexed Sso t-SNAREs (Figures
4C and
5) and interacts with the
NH2 terminus of Sso (Figure
1B), it suggests that the inhibitory effect of Vsm1 is exerted
before SNARE assembly. This interpretation agrees with a previous finding
showing that the overexpression of VSM1 affects only sec9-4,
but not sec9-7, yeast (Lustgarten
and Gerst, 1999). The Sec9-4 mutant protein is deficient in its
ability to enter into SNARE complexes at restrictive temperatures, whereas
Sec9-7 forms complexes but is unable to confer secretion
(Rossi et al., 1997).
Because both Vsm1 and Sso are evenly distributed over the plasma membrane
(Brennwald et al.,
1994; Lustgarten and Gerst,
1999), it suggests that the inhibitory action of Vsm1 (i.e.,
preventing the association of Sec9 with Sso) takes place there. Our estimates
reveal that cells have 
250,000 molecules of Sso and 120,000 of Vsm1, of
which 40% coprecipitates with Sso (our unpublished data) and 20% with Snc
(Lustgarten and Gerst, 1999).
Thus, Vsm1 is likely to regulate Sso function all over the plasma membrane and
not just at the site of exocytosis. Similar to Vsm1, Munc18 inhibits SNARE
assembly in vitro and dissociates from syntaxin after assembly occurs
(Garcia et al., 1994;
Hata et al., 1993;
Pevsner et al.,
1994). This suggests that Munc18, like Vsm1, restricts SNARE
partnering by preventing the t-SNAREs from assembling into binary complexes.
However, Sec1 in yeast is bound to SNARE complexes at the site of vesicle
fusion and may enhance their assembly (Carr
et al., 1999; Peng
and Gallwitz, 2002). Thus, Sec1 and Vsm1 seem to have very
different functions.
Overall, Vsm1 seems to be both a v- and t-SNARE regulator that controls
SNARE assembly. Because deletion of VSM1 in snc2
cells only slightly enhances their growth and has no effect upon snc
null cells (our unpublished data), we suggest that the assembly of SNARE
complexes is regulated by other, perhaps redundant, factors. This is supported
by gene knockout studies of other SNARE regulators. For example, the knockout
of synaptophysin does not significantly affect neurotransmission in mice
(McMahon et al.,
1996). In addition, deletion of LMA1 subunits in yeast does not
affect cell growth or viability (Xu et
al., 1997). Thus, some SNARE regulators either have a minor
role in SNARE assembly or are redundant with other proteins that regulate
complex formation. In conclusion, we have built upon previous findings showing
that PKA-dependent phosphorylation of t-SNAREs regulates their ability to
assemble into functional SNARE complexes
(Marash and Gerst, 2001;
Gurunathan et al.,
2002). Herein, we propose that one role for t-SNARE
phosphorylation is to recruit Vsm1, a negative regulator of secretion
(Figure 9, see model). Binding
of Vsm1 to Sso may prevent the formation of binary Sso–Sec9 SNARE
complexes, which ultimately regulates the formation of functional
trans SNARE complexes. Thus, SNARE phosphorylation adds another layer
of complexity into the regulation of membrane fusion in eukaryotes.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Footnotes |
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* Corresponding author. E-mail address: jeffrey.gerst{at}weizmann.ac.il.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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