The ArfGAP Glo3 is required for the generation of COPI vesicles

Abstract

The small GTPase Arf and coatomer (COPI) are required for the generation of retrograde transport vesicles. Arf activity is regulated by guanine exchange factors (ArfGEF) and GTPase-activating proteins (ArfGAPs). The ArfGAPs Gcs1 and Glo3 provide essential overlapping function for retrograde vesicular transport from the Golgi to the endoplasmic reticulum. We have identified Glo3 as a component of COPI vesicles. Furthermore, we find that a mutant version of the Glo3 protein exerts a negative effect on retrograde transport, even in the presence of the ArfGAP Gcs1. Finally, we present evidence supporting a role for ArfGAP protein in the generation of COPI retrograde transport vesicles.

Figure 1.

The ArfGAP Glo3, but not Gcs1, coimmunoprecipitates with the coatomer complex. (A) Protein extracts from a wild-type yeast strain were incubated with anti-Sec21 antibody, followed by incubation with protein A-agarose beads. Beads were pelleted by centrifugation, washed, and resuspended in 1× Laemmli buffer. Samples were run on a 10% SDS-PAGE, transferred to polyvinylidene difluoride membrane, and analyzed with antibodies against Gcs1 and Glo3. (B) The coimmunoprecipitation experiment was repeated in strains lacking Glo3p. The samples were analyzed as in A with antibodies against Gcs1. WCE, whole cell extract.

Figure 2.

The Glo3 ArfGAP is found on the coat of vesicles budded from Golgi membranes in vitro. Golgi membranes from wild-type cells were incubated with Arf1p, coatomer, and GTP to generate COPI vesicles. The vesicles were separated from the Golgi membranes by velocity sedimentation centrifugation. The vesicle peak was collected and floated on a buoyant density gradient. Fractions were collected, precipitated, and analyzed by immunoblot. Bet1 and Bos1 are v-SNAREs in the ER-Golgi shuttle and Emp47 is a vesicle cargo. The arrows indicate the movement of the lipid particles in the gradient.

Figure 3.

Expression of the mutant Glo3-R59K protein causes lethality. (A) Glo3-R59K-His6 was expressed in E. coli, purified using Ni²⁺/NTA-agarose, and used for an in vitro ArfGAP assay. The maximum activity observed corresponds to the hydrolysis of 82% of the total pool of radioactively labeled GTP-Arf per assay sample. Wild-type Glo3: (▪), Glo3-R59K: (•) (B) glo3Δ yeast cells harboring low-copy plasmid with the MET3pr-glo3-R59K gene were streaked for single colonies on medium containing methionine (Met+) to repress glo3-R59K expression or on medium lacking methionine (Met-) to induce glo3-R59K expression, and incubated at 30°C for 2 d. (C) Wild-type yeast cells harboring a low-copy plasmid with the MET3pr-glo3-R59K gene were streaked for single colonies on medium containing methionine (Met+) to repress glo3-R59K expression or on medium lacking methionine (Met-) to induce glo3-R59K expression, and incubated at 30°C for 2 days.

Figure 4.

Glo3-R59K coimmunoprecipitates with the coatomer complex. Protein extracts from glo3Δ mutant cells expressing Glo3-R59K were incubated with anti-Sec21 antibody, followed by incubation with protein A-agarose beads. Beads were pelleted by centrifugation, washed, and resuspended in 1× Laemmli buffer. Samples were run on a 10% SDS-PAGE and transferred to polyvinylidene difluoride membrane. The blot was decorated with Glo3 antibodies.

Figure 5.

Glo3-R59K causes accumulation of ER membrane. glo3Δ cells carrying the MET3pr-GLO3 plasmid (A), an “empty” vector plasmid (B), or the MET3pr-glo3-R59K plasmid (C) were shifted to medium lacking methionine and grown for 6 h at 30°C. Cells were collected and fixed for examination by electron microscopy. N, nucleus; Mito, mitochondria; and Vac, vacuole. The black arrow-heads point to peripheral ER in all three panels. The white arrow-heads indicate Golgi cisternae. Lipid bodies (L) are present in all strains due to growth in minimal media.

Figure 6.

Glo3-R59K causes defects in CPY maturation and secretion of the ER-resident protein Kar2. (A) Proliferating cells were exposed to [³⁵S]methionine and cysteine for 7 min and sampled without a chase with unlabeled amino acids at 0, 30, and 60 min. CPY was immunoprecipitated, resolved by SDS-PAGE, and detected by autoradiography. This panel is representative of three independent experiments. (B) Expression of MET3pr-glo3-R59K, MET3pr-GLO3, or MET3pr-empty vector was induced by incubation in medium lacking methionine for 6 h at 30°C. The medium was collected, proteins were precipitated, and analyzed by immunoblot. Kar2 was secreted only by cells expressing the ArfGAP-dead Glo3-R59K.

Figure 7.

Retrograde cargo localization requires intact Glo3 function. (A) Cells harboring the MET3pr-GLO3 or MET3pr-glo3-R59K genes were transferred to medium lacking methionine to induce gene expression. After 6 h, cells were processed for fluorescence microscopy as described previously. The retrograde cargo protein Emp47-myc was detected by Alexa 488 fluorescence (left). Cell morphology (differential interference contrast) is displayed in panels on the right. (B) Sed5-GFP is localized to the Golgi. glo3Δ cells carrying the MET3pr-GLO3 plasmid, an empty vector plasmid or the MET3pr-glo3-R59K plasmid and plasmid expressing Sed5-GFP were shifted to medium lacking methionine and grown for 6 h at 30°C. Cells were immediately examined for Sed5-GFP localization.

Figure 8.

Increased abundance of the Gcs1 ArfGAP rescues Glo3-R59K lethality. glo3Δ yeast cells harboring a low-copy plasmid with the MET3pr-glo3-R59K gene were transformed with either an empty high-copy plasmid, a high-copy plasmid carrying the GLO3 gene, or a high-copy plasmid carrying the GCS1 gene. Cells were streaked for single colonies on either Met+ or Met- media and grown at 30°C for 2 days.

Figure 9.

Glo3-R59K prevents the generation of COPI vesicles in vitro. COPI vesicles were generated from Golgi membranes isolated from yeast cells carrying the MET3pr-glo3-R59K, MET3pr-GLO3, and MET3pr-empty plasmids, which were incubated in the presence of GTP and COPI components. The vesicles were purified over a velocity gradient and subsequently floated on a Nycodenz gradient. Fractions were collected from the top, separated by SDS-PAGE, and analyzed by immunoblot. The arrows indicate the direction of movement of lipid particles within the gradient.