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. 2005 Sep 6;102(36):12777-82.
doi: 10.1073/pnas.0506156102. Epub 2005 Aug 26.

Membrane metabolism mediated by Sec14 family members influences Arf GTPase activating protein activity for transport from the trans-Golgi

Tania A Wong  1 , Gregory D Fairn, Pak P Poon, Maya Shmulevitz, Christopher R McMaster, Richard A Singer, Gerald C Johnston
Affiliations

Membrane metabolism mediated by Sec14 family members influences Arf GTPase activating protein activity for transport from the trans-Golgi

Tania A Wong et al. Proc Natl Acad Sci U S A. .

Abstract

The budding yeast Saccharomyces cerevisiae contains a family of Arf (ADP-ribosylation factor) GTPase activating protein (GAP) proteins with the Gcs1 + Age2 ArfGAP pair providing essential overlapping function for the movement of transport vesicles from the trans-Golgi network. We have generated a temperature-sensitive but stable version of the Gcs1 protein that is impaired only for trans-Golgi transport and find that deleterious effects of this enfeebled Gcs1-4 mutant protein are relieved by increased gene dosage of the gcs1-4 mutant gene itself or by the SFH2 gene (also called CSR1), encoding a phosphatidylinositol transfer protein (PITP). This effect was not seen for the SEC14 gene, encoding the founding member of the yeast PITP protein family, even though the Gcs1 and Age2 ArfGAPs are known to be downstream effectors of Sec14-mediated activity for trans-Golgi transport. Sfh2-mediated suppression of inadequate Gcs1-4 function depended on phospholipase D, whereas inadequate Gcs1-4 activity was relieved by increasing levels of diacylglycerol (DAG). Recombinant Gcs1 protein was found to bind certain phospholipids but not DAG. Our findings favor a model of Gcs1 localization through binding to specific phospholipids and activation of ArfGAP activity by DAG-mediated membrane curvature as the transport vesicle is formed. Thus, ArfGAPs are subject to both temporal and spatial regulation that is facilitated by Sfh2-mediated modulation of the lipid environment.

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Figures

Fig. 1.
Fig. 1.
The Gcs1-4 mutant protein with enfeebled ArfGAP activity is selectively impaired for transport from the trans-Golgi network. (A) Selective impairment for trans-Golgi transport. Ten-fold serial dilutions of cell populations (relevant genotype is indicated on the left) were spotted onto solid enriched medium and incubated at the indicated temperatures. The presence of a plasmid-borne WT GCS1 gene is indicated between the panels. (B) Relief of temperature sensitivity by increased ArfGAP abundance. Ten-fold serial dilutions of gcs1-4 age2Δ cell populations harboring the indicated genes on high-copy plasmids were spotted on solid defined medium for plasmid maintenance and incubated at the indicated temperatures. (C) ArfGAP activities of recombinant Gcs1 (open circles) and Gcs1-4 mutant (filled circles) proteins were assessed by in vitro hydrolysis of radiolabeled Arf-bound GTP.
Fig. 2.
Fig. 2.
Increased SFH2 and sec14-PC-only gene dosage suppresses defective Gcs1 function selectively for trans-Golgi network transport. Populations of gcs1-4 age2Δ (A) and gcs1-4 age2Δ and gcs1–28 glo3Δ (B) cells harboring the indicated genes on high-copy plasmids were treated as described in the legend of Fig. 1 A.
Fig. 3.
Fig. 3.
Gcs1-4-mediated temperature sensitivity is relieved by the presence of exogenous short-chain DAG and increased PLD activity. (A) Ten-fold serial dilutions of cells of indicated genotype (Left), carrying the WT GCS1 gene on a high-copy plasmid where indicated, were spotted on solid enriched selective medium and incubated at the indicated temperature in the presence or absence of 50 μM short-chain DAG. (B) Serial dilutions of gcs1-4 age2Δ spo14Δ triple-mutant cells harboring the indicated high-copy plasmid were treated as described in the legend of Fig. 1 A. (C) Ten-fold serial dilutions of gcs1-4 age2Δ cells harboring the indicated high-copy plasmids were treated as described in the legend of Fig. 1 A.
Fig. 4.
Fig. 4.
Exogenous DAG and overexpression of SFH2, SPO14, and SEC14-PC alleviate transport defects in gcs1-4 age2Δ cells. (A) Ratios of secreted invertase (External) to total invertase (Total) after 2 h at 37°C were determined for gcs1-4 age2Δ cells harboring the indicated genes on high-copy plasmids and also for gcs1-4 age2Δ cells growing in the presence of di8:0 DAG. Results are mean ± SD of three separate experiments, each performed in triplicate. Note that the y axis begins at a value of 0.4. (B) Endocytosis was visualized by uptake and transport of the lipophilic dye FM4-64 in gcs1-4 age2Δ cells harboring plasmids carrying the indicated genes. Samples were taken for analysis after transfer to 37°C for 30 min after the initial loading of the dye at 23°C.
Fig. 5.
Fig. 5.
Gcs1 binds to certain phospholipids. Purified recombinant Gcs1 protein (6xHis tagged) was used to probe immobilized lipids.
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