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First published online 20 January 2004
doi: 10.1242/jcs.00896

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The Arf activator Gea2p and the P-type ATPase Drs2p interact at the Golgi in Saccharomyces cerevisiae

¹ Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-5430, USA
² Departement de Biologie Joliot-Curie, CEA/Saclay, Gif-sur-Yvette F-91191, France
³ Department of Biological Sciences, Vanderbilt University, Nashville, TN 37235, USA

View larger version (45K): [in a new window]   Fig. 1. The Sec7 domain of Gea2p interacts with Drs2p. (A) Yeast two-hybrid strain Y190 carrying the C-terminal region of Drs2p (amino acids 1230-1355) in a two-hybrid prey vector and either the Gea2p Sec7 domain, full-length Gea1p or the two-hybrid bait vector alone were grown on selective medium lacking leucine and tryptophan (-L-T) that selects for each plasmid (right), or on medium containing X-gal (5-bromo-4-chloro-3-indolyl-ß-D-galactoside) (left). (B) Drs2-HA was expressed in E. coli, and cell lysates loaded onto an anti-HA antibody affinity column. The Sec7 domain of Gea2p was transcribed and translated in vitro in the presence of [35S]methionine. The radiolabeled protein was passed over the Drs2-HA column, and a control column loaded with lysates from cells not expressing Drs2-HA. (C,D) The Gea2 Sec7 domain tagged with HA was expressed in E. coli and bound to an anti-HA antibody affinity column. The full-length Drs2p C terminus, amino acids 1230-1355 (C) and two halves, amino acids 1230-1305 (D) and amino acids 1306-1355 (not shown) were radiolabeled in vitro and passed over Gea2 Sec7 domain and control columns. The bound fraction was run on SDS-PAGE gels, which were exposed to film. Coomassie Blue-stained gels of the E. coli cell lysates used to prepare each column are also shown.

View larger version (46K): [in a new window]   Fig. 2. Co-immunoprecipitation of Gea2p and Drs2p from yeast cells. C13-ABYS86 cells carrying 2µ-DRS2 (pRS425-DRS2) and either 2µ-GEA2 (pGMS3) or 2µGEA2-HA (pGMS3-HA) were grown to exponential phase and lysates prepared. Gea2p-HA was immunoprecipitated using anti-HA antibodies, and the bound material run on an SDS-polyacrylimide gel and transferred to nitrocellulose. Antibodies against Drs2p were used to probe the blots.

View larger version (42K): [in a new window]   Fig. 3. Localization of Gea2p in wild-type and drs2 cells. Wild-type CJY088 (A,C) and CYJ089 drs2 (B,D) cell membrane preparations were fractionated on OptiPrepTM gradients as described in Materials and Methods. Antibodies against different organelle markers were as follows: (A,B) Vacuole, 100 kDa subunit of the vacuolar ATPase; endosome, Pep12p; ER, Dpm1p; Golgi, Sed5p; (C,D) early Golgi, Sec22; late Golgi, Kex2p. (E) Wild-type CJY090 and (F) CJY091 drs2 cells expressing Gea2-3xGFP from its chromosomal location were grown to mid-logarithmic phase and observed by fluorescence microscopy.

View larger version (25K): [in a new window]   Fig. 4. A drs2 gea2 double mutant strain has a more severe cold-sensitive growth defect than a drs2 mutant alone. Strains SEY6210 (WT), CJY049-2-1 (gea2), SEY6210 drs2::URA3 (drs2) and SCY020-9-4 (drs2 gea2) alone or carrying the indicated plasmids, were grown to stationary phase, and serial dilutions were spotted onto plates. Note that drs2 gea2 fails to grow at 27°C, a temperature at which drs2 grows well.

View larger version (35K): [in a new window]   Fig. 5. The C-terminal region of Drs2p is essential for its function. (A) Sequence alignment of the entire C-terminal cytoplasmic region of Saccharomyces cerevisiae (Sc) Drs2p (accession number NP_009376) and its homologues from Ajellomyces capsulatus (accession number AAF90186) and Schizosaccharomyces pombe (accession number NP_596486). Also shown is an alignment of a region conserved between mammalian and yeast ATPases. (B) C-terminal truncations and internal deletions of Drs2p were transformed into SEY6210 drs2::TRP1, and ability to grow at 20°C was determined. Truncations or deletion mutants that failed to grow at 20°C were transformed with multicopy plasmids carrying GEA1 and GEA2, and growth was monitored at 20°C. In each case, overexpression of either Gea1p or Gea2p partially suppressed the growth defect. Hatched box, Gea2p interaction region; black box, highly conserved region; underlined, NPFXD motifs. (C) SEY6210 drs2::TRP1 was co-transformed with low-copy centromeric plasmids carrying drs2-D560N and the C-terminal truncation mutant drs21246-1355 (lower panel), and appropriate vector controls (upper panel). Some variability in the level of growth of the four independently isolated drs2-D560N + drs21246-1355 co-transformants at the non-permissive temperature was noted, but in all cases significant growth, compared to a drs2 strain, was evident.

View larger version (35K): [in a new window]   Fig. 6. The gea2-V698G mutant fails to interact with Drs2p, and is temperature and BFA-sensitive. (A) Strain Y190 carrying the C-terminal region of Drs2p (amino acids 1230-1355) in the two-hybrid prey plasmid and either the wild-type Gea2 or gea2-V698G mutant Sec7 domain as the bait plasmid were grown on medium containing X-gal (left) or on control medium (right). (B) Strains CJY092 gea1 gea2/pSKP1 (pCEN-GEA2) and CJY093 gea1 gea2/pCEN-gea2-V698G were grown to stationary phase and serial dilutions spotted onto plates grown either at 30°C or 37°C. (C) Strains CJY092 gea1 gea2/pSKP1(pCEN-GEA2) and CJY093 gea1 gea2/pCEN-gea2-V698G were grown to stationary phase and serial dilutions spotted onto plates containing 30 or 50 µg/ml BFA or solvent alone (methanol). Plates were incubated at 30°C.

View larger version (38K): [in a new window]   Fig. 7. Trafficking phenotype of the gea2-V698G mutant. Strains CJY092 gea1gea2/pSKP1(pCEN GEA2) and CJY093 gea1gea2/pCEN-gea2-V698G were grown to exponential phase and incubated for 1 hour at 38°C, pulse-labeled for 10 minutes with [35S]methionine/cysteine, then chased with non-radioactive amino acids for 30 minutes, all at 38°C. (A) Proteins secreted into the medium were recovered and analyzed as described in Materials and Methods. Strains CJY062-10-3 gea1-4 gea2 and APY022 gea1-6 gea2 (Peyroche et al., 2001) were treated similarly and included as controls. (B) Strains listed above were grown at 30°C and shifted to the indicated temperature for 1 hour before labeling for 5 minutes. Cell lysates were prepared at the chase times indicated and immunoprecipitated with anti-CPY antibodies. The ER-early-Golgi localized p1 precursor, late-Golgi p2 precursor, and mature (m) forms of CPY are indicated. Note that at 20°C, transport through the secretory pathway is significantly slowed, and no mature CPY accumulates during the short time course of this experiment.

View larger version (157K): [in a new window]   Fig. 8. Ultrastructural analysis of gea2-V698G and control cells grown to exponential phase at 24°C, then shifted for 1 hour to 38°C. Stereopairs of 0.20 µm thick sections of (A,B) CJY092 gea1 gea2/pCEN-GEA2 and (C-F) CJY093 gea1 gea2/pCEN-gea2-V698G cells. ER, endoplasmic reticulum, G, Golgi, V, vacuole, SG, secretion granule/vesicle. (A) An image showing typical Golgi tubular network structures (G) scattered throughout the cytoplasm. (B) A cell with an emerging bud (lower right) with a typical accumulation of homogeneously sized secretion granules/vesicles (SG). (C) Cell showing abnormal accumulation of membranes throughout the cell. (D) Cell with an emerging bud lacking the normal accumulation of secretion granules/vesicles. Note the heterogeneous size and scattered distribution of secretion granules/vesicles (arrowheads). To view the 3D image of the structures, figures should be viewed through a pair of anaglyph (red-cyan) glasses with the red lens placed over the left eye. Scale bars: 0.5 µm.

View larger version (150K): [in a new window]   Fig. 9. (A,B) Stereopairs of 0.20 µm thick sections of CJY093 gea1 gea2/pCEN-gea2-V698G cells prepared as described in Fig. 8. (A) Cell showing an abnormal Golgi element (arrow) with heterogeneously sized fenestrations and segregating granules (one indicated with an arrowhead). (B) Cell showing an abnormal Golgi element with almost no fenestrations (arrow) but nevertheless containing a segregating granule (arrowhead). (C-E) Strain SEY6210 drs2URA3 was grown to exponential phase at 30°C, then shifted to 24°C for 1 hour. Stereopairs of 0.20 µm thick sections are shown. (C) Cell showing accumulation of spherical membrane structures (S) made up of unfenestrated membrane. (D) A higher magnification image of a spherical element made up of abnormally fenestrated membrane (white arrowhead). The element is connected to a ribbon of ER that, on the right, is continuous with the nuclear envelope. (E) Cell showing an abnormal Golgi element with almost no fenestrations (arrow) and containing segregating granules (one is marked by an arrowhead). ER, endoplasmic reticulum; G, Golgi; V, vacuole; SG, secretion granule/vesicle; S, spherical structure. To view the 3D image of the structures, figures should be viewed through a pair of anaglyph (red-cyan) glasses with the red lens placed over the left eye. Scale bars: 0.5 µm.

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