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. 2013 Dec 10;110(50):19995-20002.
doi: 10.1073/pnas.1320029110. Epub 2013 Nov 18.

Phosphorylation of the Rab exchange factor Sec2p directs a switch in regulatory binding partners

Danièle Stalder  1 , Emi Mizuno-Yamasaki, Majid Ghassemian, Peter J Novick
Affiliations

Phosphorylation of the Rab exchange factor Sec2p directs a switch in regulatory binding partners

Danièle Stalder et al. Proc Natl Acad Sci U S A. .

Abstract

Sec2p is a guanine nucleotide exchange factor that promotes exocytosis by activating the Rab GTPase Sec4p. Sec2p is highly phosphorylated, and we have explored the role of phosphorylation in the regulation of its function. We have identified three phosphosites and demonstrate that phosphorylation regulates the interaction of Sec2p with its binding partners Ypt32p, Sec15p, and phosphatidyl-inositol-4-phosphate. In its nonphosphorylated form, Sec2p binds preferentially to the upstream Rab, Ypt32p-GTP, thus forming a Rab guanine nucleotide exchange factor cascade that leads to the activation of the downstream Rab, Sec4p. The nonphosphorylated form of Sec2p also binds to the Golgi-associated phosphatidyl-inositol-4-phosphate, which works in concert with Ypt32p-GTP to recruit Sec2p to Golgi-derived secretory vesicles. In contrast, the phosphorylated form of Sec2p binds preferentially to Sec15p, a downstream effector of Sec4p and a component of the exocyst tethering complex, thus forming a positive-feedback loop that prepares the secretory vesicle for fusion with the plasma membrane. Our results suggest that the phosphorylation state of Sec2p can direct a switch in its regulatory binding partners that facilitates maturation of the secretory vesicle and helps to promote the directionality of vesicular transport.

Keywords: membrane traffic; phospho-regulation; vesicle maturation; yeast.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Phosphorylation of Sec2p increases binding to the exocyst component Sec15p in vitro. (Left) GST-Sec2p was purified from bacteria (Sec2b) or from yeast (Sec2y). Sec2y was treated with the phosphatase CIP or was left untreated. His6-Sec15p was purified from bacteria, eluted, and incubated with GST-Sec2p immobilized on glutathione beads. Bound Sec15p was detected with anti-Sec15p antibody, and GST-Sec2p was detected with anti-GST antibody. (Right) The percentage of bound Sec15p was calculated and is indicated. The mean and SD of three different experiments are shown. *P < 0.002; **P < 0.05, Student t test.
Fig. 2.
Fig. 2.
Identification of three previously unknown phosphosites in the Sec15p/Ypt32p-binding region. (A) Domain organization of Sec2p. The N-terminal region contains a coiled-coil domain (CC) that catalyzes the exchange of GDP for GTP on Sec4p. Just downstream, are overlapping binding sites for Sec15p and Ypt32p-GTP, which compete against each other to bind to Sec2p. Three positively charged patches allow Sec2p to interact directly with the phosphoinositide PI(4)P. Finally, the region from amino acid 450–508 negatively regulates Sec15p binding to Sec2p by an auto-inhibitory mechanism. (B) Alignment (by Lalign, www.ch.embnet.org/software/LALIGN_form.html) of the sequence containing the serines 181, 186, and 188 with the mammalian homolog Rabin8. (C) The phosphorylation-dependent mobility shift of the nonphosphorylatable Sec2p mutant S181–8A and the phosphomimetic Sec2p mutant S181–8D/E. GST-tagged Sec2p was purified from bacteria (Sec2b) or from yeast (Sec2y) and was treated with the phosphatase CIP or was left untreated. Sec2 proteins were visualized by Coomassie Brilliant Blue staining after SDS/PAGE.
Fig. 3.
Fig. 3.
Phosphomimetic mutation of serines 186 and 188, but not 181, leads to increased binding of Sec15p and a corresponding decrease in binding of Ypt32p-GTP. (A–C) GST-Sec2p, His6-Sec15p, and His6-Ypt32p were purified from bacteria. Sec15p and Ypt32p were eluted. Ypt32p was preloaded with GTPγs and then was incubated with GST-Sec2p immobilized on glutathione beads. Bound proteins were detected with anti-Sec15p antibody or anti-Ypt32p antibody. GST-Sec2 was detected with anti-GST antibody.
Fig. 4.
Fig. 4.
A Sec2p phosphomimetic allele in which the entire S/T patch was mutated displays increased binding of Sec15p in vitro and in vivo. (A) In vitro binding assays, performed as in Fig. 3, of bacterial purified wild-type GST-Sec2p, S181–8A, and S181–8D/E proteins with His6-Sec15p (Upper; the vertical white space indicates where intervening lanes have been removed for presentation purposes) or His6-Ypt32p-GTPγs (Lower). (B) Phosphomimetic Sec2p mutant S181–8D/E has an enhanced interaction with Sec15p in yeast cells. Wild-type Sec2p-3×GFP, S181–8A, or S181–8D/E was immunoprecipitated with GFP antibody in yeast strains coexpressing Sec15p-13×myc (NY3047, NY3048, and NY3049, respectively). A yeast strain expressing untagged Sec2p (NY2546) was used as a negative control. Coprecipitated Sec15p-13×myc and the amount of Sec15p-13×myc in 0.2% of the lysates were detected with anti-myc antibody. The amount of Sec2p-3×GFP in the immunoprecipitates was detected with anti-GFP antibody. The intensity of the bands was quantified using ImageJ. The percentage of Sec15p bound to Sec2p was calculated and is indicated. The mean and SD of three different experiments are shown. *P < 0.03, Student t test.
Fig. 5.
Fig. 5.
Phosphomimetic Sec2 mutation inhibits binding to the phosphoinositide PI(4)P. Bacterial purified wild-type GST-Sec2p, Sec2p KA (negative control), and Sec2p with phospho-mutations (S181–8A or D/E) were incubated with liposomes containing, or not, 5 mol% PI(4)P. Liposomes were precipitated, and proteins in the supernatant (S) and in the pellet (P) were visualized by Coomassie Brilliant Blue staining after SDS/PAGE. The intensity of the bands was quantified using ImageJ. The mean and SD of three different experiments are shown. *P < 0.05, Student t test.
Fig. 6.
Fig. 6.
Locking Sec2p in either its phosphorylated or unphosphorylated state affects both its localization and yeast growth. (A) Tetrads dissected from diploid strains carrying the sec2–3×GFP S181–8A or D/E, ypt31Δ, and ypt32A141D alleles. ypt31Δ and ypt32A141D spores are shown within squares. The triple-mutant spores ypt31Δ and ypt32A141D sec2p S181–8A or D/E are shown within continuous or dashed circles, respectively. (B) Serial dilutions of cells expressing different phospho-mutant alleles of Sec2p in the ypt31Δ ypt32A141D background. Cells were spotted and grown on YPD plates at 25 °C, 30 °C, or 32 °C for 2 d or at 16 °C 6 d. (C) Localization of wild-type Sec2p-3×GFP and phospho-mutants (S181–8A and D/E) fused to 3×GFP. Cells were grown overnight at 25 °C to midlog phase in a synthetic medium containing 2% glucose and then were pelleted and observed immediately. (Scale bar, 10 μm.) Around 150 cells were counted for each experiment, and the mean and SD of three different experiments are shown. Values indicate the percentage of cells showing a polarized localization of Sec2p in unbudded cells or an accumulation of Sec2p at the bud tip of small/medium cells or at the mother–daughter neck of larger cells. *P < 0.02; **P < 0.005, Student t test.
Fig. 7.
Fig. 7.
A nonphosphorylatable Sec2 mutant affects vesicular export from the Golgi. Triple mutants ypt31Δ ypt32A141D sec2-3×GFP [either wild-type (NY3040) or S181–8A (NY3045)] were shifted at midlog phase to 32 °C for 1 h and were processed for thin-section electron microscopy.
Fig. 8.
Fig. 8.
A model for the Sec2p phospho-cycle in the secretory pathway. Sec2p, in its nonphosphorylated form, is recruited to the Golgi membrane by binding to both Ypt32p-GTP and PI(4)P. The Golgi-derived secretory vesicle pinches off, and Sec2p activates Sec4p, which then recruits its effector, Sec15p. Before delivery of the vesicle to the plasma membrane, the level of PI(4)P decreases, triggering a conformation change in Sec2p, which allows Sec15p to replace Ypt32p-GTP on Sec2p. Sec2p undergoes phosphorylation that enhances the interaction of Sec2p with Sec15p and inhibits binding to Ypt32p-GTP and PI(4)P, pushing the reaction forward. This process creates a positive-feedback loop because the GEF Sec2p is able to bind directly to an effector of the activated Rab Sec4p. Thus, a microdomain of high Sec4p-GTP and high effector concentration is generated, facilitating the delivery, tethering, and fusion of the vesicle with the plasma membrane. Sec2p then is dephosphorylated, triggering its release from Sec15p and allowing Sec2p to associate with a new round of secretory vesicles, thus ensuring the continuity of vesicular transport.
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