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The ARF exchange factors Gea1p and Gea2p regulate Golgi structure and function in yeast

¹ Service de Biochimie et Génétique Moléculaire, Bat. 142, Département de Biologie Cellulaire et Moléculaire, CEA/Saclay, 91191 Gif-sur-Yvette, France
² Cell Biology and Metabolism Branch, NICHD, NIH, Bldg. 18T, Room 101, 18 Library Drive, Bethesda, MD 20892-5430, USA

View larger version (1K): [in a new window]   Fig. 1. Positions of the amino acid substitutions in the gea1-4, gea1-6 and gea1-19 alleles. Sequence alignments (with three other ARF GEFs) of the regions of Gea1p in which these changes occur are indicated. GenBank accession numbers for the sequences shown are: Gea1p (Z49531); Gnom/Emb30 (U36433); C. elegans ARF GEF (Z81475); and GBF1 (AF068755). The Sec7 domain of Gea1p is indicated by a striped box. gea1-4 has additional substitutions in the C-terminal portion of the protein downstream of the Sec7 domain, but these are in poorly conserved regions of the protein. gea1-6 has only the two substitutions shown. gea1-19 has two additional substitutions, but only those in regions of clearly defined homology among the four ARF GEFs shown are indicated. Note that gea1-6 and gea1-19 both have one substitution, L862S and M859K, respectively, in the same well-conserved region. Identities in the sequence alignment are boxed, and similarities are shaded.

View larger version (58K): [in a new window]   Fig. 2. Secretion into the medium in gea mutants. (A) WT (CJY049-2-3), gea1-4 (CJY054), sec18-1 (RSY271) and sec21-3 (EGY1213) strains were preincubated at 38°C for 45 minutes, pulse-labeled for 10 minutes with [35S]methionine/cysteine, then chased with non-radioactive amino acids for 30 minutes at 38°C. Proteins secreted into the medium were recovered as described in Materials and Methods, and analyzed by SDS-PAGE and fluorography. Filled circles, proteins that continue to be secreted in sec21-3 and gea mutants; arrowheads, proteins whose secretion is blocked or severely retarded. (B) Wild-type strain CJY049-11-1 GEA1 and the mutant APY026 gea1-19 were preincubated for 20 minutes at 37°C, then pulse-labeled for 10 minutes and chased for the indicated times at 37°C. Proteins secreted into the medium were recovered and analyzed. (C) Strains CJY049-2-3 GEA1 and CJY054 gea1-4 were preincubated at 38°C for the times indicated, then pulse-labeled for 10 minutes and chased for 5 minutes before analyzing proteins secreted into the medium. (D,E) Strains CJY52-10-2/pCLJ90-GEA1 and CJY052-10-2/pNTS6-gea1-6 (D) and strains CJY52-10-2/pCLJ90-GEA1 and CJY052-10-2/pNTS19-gea1-19 (E) were preincubated at 37°C for the times indicated, then pulse-labeled for 10 minutes and chased for 20 minutes (D) or 30 minutes (E) prior to recovery of medium-secreted proteins.

View larger version (30K): [in a new window]   Fig. 3. (A) Strains CJY49-2-3 and CJY054 were preincubated for 45 minutes at 38°C, labeled for 10 minutes and chased for 30 minutes, all at 38°C. Cells and medium were separated and HSP150 immunoprecipitated using polyclonal anti-HSP150 antiserum. (B) Strains CJY52-10-2/pCLJ90-GEA1 and CJY052-10-2/pNTS19-gea1-19 were preincubated for 30 minutes at 37°C, labeled for 10 minutes and chased for 10 minutes, all at 37°C. Cells were then converted to spheroplasts, and internal and external fractions immunoprecipitated with antibodies against invertase. (C,D) Strains CJY52-10-2/pCLJ90-GEA1 and CJY052-10-2/pNTS19-gea1-19 were preincubated for 30 minutes at 37°C, labeled for 10 minutes and chased for the amount of time indicated. Cell extracts were immunoprecipitated with anti-ALP1 monoclonal antibodies (C) or with anti--pheromone antiserum (D), and immunoprecipitates analyzed by SDS-PAGE.

View larger version (45K): [in a new window]   Fig. 4. CPY transport and maturation were monitored by pulse-chase analysis. (A) Strain APY026 gea1-19 and wild-type strain CJY049-11-1 were incubated at 37°C for 30 minutes, pulse-labeled for 10 minutes, then chased for the times indicated. CPY was recovered from cell extracts by immunoprecipitation and visualized by SDS-PAGE and fluorography. (B) A pulse-chase regime identical to that described in part (A) was carried out for APY022 gea1-6 and the corresponding wild-type strain (CJY049-3-4), followed by two successive immunoprecipitations, first using anti-CPY, then anti-1,6-antisera. (C) Strain CJY062-10-3 gea1-4 and CJY049-3-4 (wildtype) were subjected to a pulse-chase regime as described in part (A) except that preincubation was carried out at 38°C for 30 minutes. Single CPY immunoprecipitations were carried out. (D) After a first immunoprecipitation with anti-CPY antibodies as described in part (A) for strains APY026 gea1-19 and CJY049-11-1 (GEA1), a subsequent immunoprecipitation was carried out using anti-1,6-antiserum. ‘mCPY’ refers to the mature vacuolar form; ‘p’ indicates a precursor form; p1 is the ER-core-glycosylated precursor; p2 is the fully glycosylated Golgi form; p and pß are ER precursor forms because they do not contain 1,6-linkages. ‘p1*’ is a precursor form found in the gea1 mutants that does not correspond to p1, p2 or mCPY as seen in a wild-type strain; it is a Golgi form since it is precipitated by anti-1,6-antiserum. (E) Percentages of precursor forms (all forms grouped) and of mature vacuolar CPY for wildtype and each gea1 mutant, as determined by quantitative Phosphorimager analysis. The values obtained for the amount of mCPY for the wild-type strain in three separate experiments were 64%, 67% and 69%: the average is shown.

View larger version (23K): [in a new window]   Fig. 5. Localization of Och1-HA in wildtype and gea mutant strains. The wild-type strain CJY49-3-4 (A), strain CJY62-10-3 gea1-4 (B) and strain APY022 gea1-6 (C), each carrying plasmid pOH (Och1-HA), were grown at 24°C, and either shifted to 37°C for 40 minutes, or left at 24°C. Cells were fixed and prepared for immunofluorescence analysis using monoclonal anti-HA antibodies. Left panels, DAPI staining to visualize nuclei; right panels, anti-HA. Arrows indicate ring-like structures and arrowheads ER/nuclear envelope staining. Bar, 5 µm.

View larger version (35K): [in a new window]   Fig. 6. Localization of Anp1p and Och1-HA in gea mutants. Strains CJY62-10-3 gea1-4/pOH (A) and APY022 gea1-6/pOH (B) were grown at 24°C, and either shifted to 37°C for 40 minutes, or left at 24°C. Cells were fixed and prepared for immunofluorescence analysis using rabbit affinity-purified anti-Anp1p antiserum and mouse monoclonal anti-HA antibodies. Arrows indicate ring-like structures. Bar, 5 µm.

View larger version (47K): [in a new window]   Fig. 7. Localization of Mnn1-Myc and Och1-HA in gea1-4. Strain CJY62-10-3 gea1-4/pOH was grown at 24°C (A) or at 37°C for 15 minutes (B) or 40 minutes (C). Cells were fixed and prepared for immunofluorescence using rabbit anti-HA antibodies and mouse anti-Myc monoclonal antibodies. Arrows point to the ring-like structures that contain both Mnn1-Myc and Och1-HA. Bar, 5 µm.

View larger version (34K): [in a new window]   Fig. 8 Strains CJY49-3-4 (wildtype), CJY62-10-3 gea1-4 and APY022 gea1-6 were grown at 24°C, then shifted to 37°C for 10 minutes. Cells were spun down, resuspended in a small volume of medium prewarmed to 37°C, and FM4-64 added at time 0. Cells were incubated at 37°C, and at the time points shown (minutes after addition of FM4-64), cells were removed and fixed with formaldehyde. Bar, 5 µm.

View larger version (144K): [in a new window]   Fig. 9. Stereopairs of 0.20 µm thick sections of gea1-4 mutant cells incubated at 37°C for 40 minutes. (A) A curved tubular network structure (arrow) is connected to an ER sheet (ER; top-left). The central portion consists of a network of wide-meshed, lightly-stained tubules, and at the periphery, larger and more intensely stained tubules display nodules (arrowhead) adjacent to secretion granules (SG). Magnification x73,500. (B) A large tubular network (arrow) with small dilations at the intersections of polygonal meshes (arrowheads) forms a spherical mass and is connected to an ER ribbon continuous with a sheet of subplasmalemmal ER (ER). N, nucleus. Magnification x74,500. (C) Tubular networks form ring-like structures (arrows). Wide polygonal meshes are clearly visible in the upper structure, seen in oblique view. Below, fenestrated and tubular structures seen in profile form two concentric rings. SG, secretion granule. Magnification x87,500. (D) A large, relatively flattened tubular network consists of lightly-stained, wide polygonal meshes (arrow) with a few small dilations (arrowhead). SG, secretion granule; V, vacuole. Magnification x70,500. (E) A tubular network (arrow, top-left) with darkly stained dilations (arrowhead) forms a ring in proximity to the nuclear envelope. At the bottom-right another tubular network forms a large ovoid mass (arrow). Two ER sheets in continuity with the nuclear envelope are labeled ER. N, nucleus. Magnification x82,300.

View larger version (158K): [in a new window]   Fig. 10. Stereopairs of 0.20 µm thick sections of gea1-6 mutant cells incubated for 40 minutes at 37°C. (A) Non-fenestrated ER sheets accumulate to form a broad network (ER) interconnecting the nuclear envelope and the subplasmalemmal ER. V, vacuole; N nucleus. Magnification x36,200. (B) Cylinders or spheres of unfenestrated membrane (arrowheads) are dispersed throughout the cytoplasm. Note the absence of tubular networks and secretory granules. N, nucleus. Magnification x35,600. (C) A small tubular network (arrow) is continuous on one side with an ER sheet seen in profile, and is continuous on the other side with a multi-layered cylindrical structure (arrowheads). A sheet of unfenestrated ER (ER) is seen in oblique view (bottom-right). N, nucleus. Magnification x55,600. (D) A cylindrical structure is made up of anastomosed ribbons of unfenestrated membrane (arrowheads). V, vacuole. Magnification x57,700.