The yeast p24 complex is required for the formation of COPI retrograde transport vesicles from the Golgi apparatus

Abstract

The p24 family members are transmembrane proteins assembled into heteromeric complexes that continuously cycle between the ER and the Golgi apparatus. These cargo proteins were assumed to play a structural role in COPI budding because of their major presence in mammalian COPI vesicles. However, this putative function has not been proved conclusively so far. Furthermore, deletion of all eight yeast p24 family members does not produce severe transport phenotypes, suggesting that the p24 complex is not essential for COPI function. In this paper we provide direct evidence that the yeast p24 complex plays an active role in retrograde transport from Golgi to ER by facilitating the formation of COPI-coated vesicles. Therefore, our results demonstrate that p24 proteins are important for vesicle formation instead of simply being a passive traveler, supporting the model in which cargo together with a small GTPase of the ARF superfamily and coat subunits act as primer for vesicle formation.

Figure 1.

The emp24Δ and erv25Δ mutations interact genetically with mutant alleles of genes encoding for COPI vesicle coat components. (A) The emp24Δ glo3Δ strain with the URA3 based plasmid bearing GLO3 pMMY63 was transformed with LEU2-based empty vector pRS315 or GLO3 encoding plasmid pMMY67. Double transformants were replica plated at 30°C on SD-leu-ura or on 5-FOA medium to induce loss of pMMY63. (B) The emp24Δ or erv25Δ strains were crossed with a gcs1Δ strain and the resulting haploid spores were tested for growth at 30°C on YPUAD. (C) emp24Δ or erv25Δ strains were crossed with a sec21-1 strain and the resulting haploid spores were tested for growth at 24 and 30°C and grown on YPUAD.

Figure 2.

The emp24Δ ret4-1 double mutant cells exhibit COPI mutant–like phenotypes. (A) emp24Δ strain was crossed with a ret4-1 strain and the resulting haploid spores were tested for growth at 24 and 35°C on YPUAD. (B) Proliferating cells were radiolabeled for 5 min, chased for 30 min at 24°C, and lysed. CPY was immunoprecipitated, resolved by SDS-PAGE, and analyzed by PhosphoImager. ER (p1), Golgi (p2), and vacuole (m) CPY forms are indicated. (C) Low glucose–induced cells were pulse-labeled for 5 min and chased for 30 min at 24°C. Cells were converted to spheroplasts, and separated into intracellular (I) and extracellular (E) fractions from which invertase was immunoprecipitated, resolved by SDS-PAGE, and analyzed by PhosphoImager. Migration positions of core glycosylated, hypo-, and hyperglycosylated invertase are indicated. (D) Cells without (1, 2, 3, and 4) or with (5, 6, 7, and 8) ERD2 2μ plasmid (pJS209) were transferred to fresh media for 1 h at 24°C. Proteins contained in the cell culture supernatant were concentrated by TCA precipitation, resolved by SDS–PAGE, and Kar2p and Hexokinase were detected by immunoblotting. (E) β-Galactosidase assays were performed on strains harboring the reporter construct (pJC31). (F) Fluorescence images of cells expressing the ER-localized protein GFP-HDEL. Cells were grown at 24°C, except sec21-1 cells, which were grown at 24°C and then shifted 40 min at 37°C. Bar, 5 μm.

Figure 3.

Golgi membranes derived from emp24Δ mutant cells are defective for the generation of COPI vesicles in vitro. Vesicles were generated from wild-type and emp24Δ (RH4443) Golgi membranes, which were incubated with GTP in the absence (A) or the presence of an excess of COPI components (B). The vesicles were purified over a velocity gradient, and subsequently floated on a Nycodenz gradient. Vesicle-containing fractions were collected and pooled, TCA precipitated, resolved on SDS-PAGE, and analyzed by immunoblot using antibody against Emp47p.

Figure 4.

Specific genetic and physical interactions between p24 members and COPI coat components. (A) The emp24Δ sec21-1 and erv25Δ sec21-1 cells were transformed with empty plasmid (YEp352) or a high-copy plasmid carrying either the GLO3 (pPPL43) or the GCS1 gene (pPP421). Transformed cells were tested for growth at 24 and 30°C. (B and C) Synthetic peptides corresponding to cytoplasmic domains of Emp24p (RRFFEVTSLV) and Erv25p (KNYFKTKHII) were coupled to thiopropyl-Sepharose beads and incubated with cytosol (B) or recombinant Glo3p (C). Bound material was resolved by SDS-PAGE and analyzed by immunoblot using antibodies against Glo3p and Gcs1p.

Figure 5.

UPR compensates the loss of retrograde transport function in the absence of p24 proteins. Cells expressing Rer1p-GFP were observed by fluorescence microscopy at 24°C, or after the shift to 37°C for 20 min. Bar, 5 μm.