YSL2 encodes a novel protein with homology to Sec7 family GEFs. (A) Alignment of the putative Sec7 domain from Ysl2p/YN37_YEAST (identifier in boldface red) between positions 220 and 504 with 24 other Sec7 domains, as defined by the SMART database (44). (B) Alignment of the N-terminal region of Ysl2p/YN37_YEAST (amino acids 1 to 175) with five other Sec7 family members from mammals (Q9Y6D6/BIG1 and Q9Y6D5/BIG2), worms (Q19338 and Q9XWG5), and fission yeast (YAED_SCHPO). All sequence identifiers are taken from the SWALL database. Amino acid symbols are coded and shaded by property: yellow with gray shading, highly conserved; others are indicated in parentheses in the following consensus description. For the consensus sequence, the following symbols are used: +, positive (blue); −, negative (red); ∗, Ser/Thr (cyan); l, aliphatic (gray); t, tiny (green); a, aromatic (blue); c, charged (magenta); s, small (green); p, polar (blue); b, big (blue with light gray shading); h, hydrophobic (black with yellow shading); s, default.

The temperature-sensitive ysl2-307 and ysl2-316 mutants exhibit vacuole fragmentation rapidly upon a shift to the nonpermissive temperature and are specifically defective in the degradation of Ste2p at the permissive temperature. (A) Growth of wild-type (wt; BS1019) cells, strains carrying temperature-sensitive alleles ysl2-307 (BS1020) and ysl2-316 (BS1021), and the Δysl2 strain (BS747 carrying the empty YCp50 vector). Cells were streaked out on YPD and incubated at 25 and 37°C. (B) LY internalization assays either were directly performed at 30°C with cells described in the panel A legend or were performed with cells that were incubated at 37°C for 20 min prior to the 1-h incubation with LY at 30°C. After being washed, cells were visualized by using fluorescence (left) and Nomarski optics (right). Bar, 10 μm. (C) RH1201 (wild type) and BS1021 (ysl2-316) were grown in YPD at 25°C to early log phase. Cells were either directly cryoimmobilized and processed for electron microscopy (EM) as described in Materials and Methods or shifted to 37°C for 30 min, quick-frozen, and processed for EM analysis. Thin sections were viewed in the electron microscope. N, nucleus. (D) α-Factor-induced internalization of Ste2p was analyzed on cells (BS1022, BS1023, and BS1024) at 30°C as described for Fig. 2A. ∗, band specifically labeled by the anti-Ste2p serum. (E) Invertase secretion was performed with cells listed in the panel D legend, which were grown in YPD (5% glucose) at 25°C. Invertase derepression was induced in YPD (0.1% glucose) at 30°C for 30 min. Subsequently the cells were either left at 30°C or shifted to 37°C. Samples were analyzed for external and total invertase activity after 0-, 50-, 60-, and 70-min periods of invertase derepression. The values represent means of three different experiments.

GFP-Ysl2p is localized to punctate structures. (A and B) BS819 (homozygous diploid for YSL2::GFP) (A) and RH1201 (B) cells were processed for fluorescence microscopy as described in Materials and Methods. Images were obtained by fluorescence (left) and Nomarski optics (right). Bar (applies to all panels), 10 μm. (C) After BS819 cells were labeled with FM4-64 for 30 min at 0°C, cells were resuspended in SD prewarmed to 15°C to allow the accumulation of FM4-64 during a 30-min incubation. Subsequently, cells were collected, resuspended in fresh medium, and photographed within 2 min with appropriate filters for GFP (left) and FM4-64 (right) fluorescence. (D) BS819 cells were incubated with FM4-64 as described for panel C with the difference that FM4-64 was internalized at 30°C for 30 min to allow transport of FM4-64 to the vacuole. (E to G) BS819 (wild type; E), BS953 (vps27; F), and BS817 (sec7; G) cells were grown at 25°C. BS819 and BS953 cells were directly used to analyze the pattern of GFP-Ysl2p fluorescence as described for panel A. BS817 (sec7) cells were harvested, resuspended in YP medium prewarmed to 37°C containing 0.1% glucose, and incubated at 37°C for 2 h to induce the Golgi defect caused by mutant sec7. Subsequently, cells were viewed as described for panel A.

Ysl2p binds to Arl1p via its Sec7 and N-terminal domains. (A) The Sec7 domain of Ysl2p interacts with Arl1p in the two-hybrid system. Strain Y190 was cotransformed with combinations of pAS1-Ysl2Sec7 and pACTII-Arl1wt, pACTII-18aaArl1^T32N, pACTII-18aaArl1^G30A, or pACTII-18aaArl1^Q72L and pACTII-Ysl2Sec7 and pAS1-Ypt51^S21N. Transformants were streaked out on SD-Leu, -Trp, and -His plates containing 25 mM 3-amino-1,2,4-triazole and analyzed for β-galactosidase activity as described in Materials and Methods. The values are the averages of three to five independent experiments with triplicate assays per transformant. Error bars, standard deviations. wt, wild type. (B) GST-Ysl2Sec7 and GST-Ysl2Nterm bind to in vitro-translated Arl1^T32N. Recombinant GST (10 μg; lane 1 and 6), GST-Ysl2Nterm (9.6, 19.2, and 38.4 μg in lanes 2 to 4 and 7 to 9, respectively), and GST-Ysl2Sec7 (30 μg; lanes 5 and 10) were purified from BL21 cells and immobilized onto glutathione-Sepharose beads. After incubation with ³⁵S-labeled in vitro-translated Arl1^T32N or −18aaArl1^T32N, the beads were washed and bound proteins were eluted by boiling them in SDS-PAGE sample buffer and separated by SDS-PAGE. Gels were either stained with Coomassie brilliant blue (Co) to reveal the GST fusions or processed for autoradiography (Au) to visualize Arl1^T32N and −18aaArl1^T32N. Quantitation of bound Arl1^T32N and −18aaArl1^T32N was performed with the phosphorimager. ∗1, full-length GST-Ysl2Nterm; ∗2, full-length GST-Ysl2Sec7.

The Δysl2 mutant is blocked in the degradation of Ste2p and contains highly fragmented vacuoles. (A) Pheromone-stimulated internalization of Ste2p was analyzed on cells (BS64 and BS694) grown at 25°C. After a 10-min preincubation in the presence of 10 μg of cycloheximide/ml, α-factor was added and, at the indicated times, samples were withdrawn and processed for immunoblotting with an anti-Ste2p antiserum. A cell extract of α-mating-type cells lacking Ste2p is shown (α cell, BS188) to reveal a band (∗) unspecifically labeled by the anti-Ste2p serum. The positions of the 45- to 47-kDa and lower-mobility forms of Ste2p (hp+u) are indicated. wt, wild type. (B) LY internalization assays were performed with cells (RH1201 and BS747) grown at 25°C. After a 1-h incubation with LY at 30°C, the cells were washed, mounted in low-melting-point agarose, and visualized by using fluorescence (right) and Nomarski optics (left). Bar, 10 μm.

Ysl2p is highly enriched in a membrane pellet fraction produced by centrifugation at 100,000 × g and cofractionates with endosomal membranes upon flotation into a Nycodenz gradient. (A) Wild-type cells (RH732) were converted to spheroplasts and lysed with DEAE-dextran. The precleared lysate was subjected to differential centrifugation at 3,500 × g, 13,000 × g, and 100,000 × g to generate pellet fractions P1, P2, and P3 and supernatant fraction S3. Immunoblot analysis was performed with antibodies against Ysl2p and marker proteins for ALP (vacuole), porin (mitochondria), phosphoglycerate kinase (cytosol), Sec61p (ER), and Vps10p (late Golgi). (B) The P3 fraction pellet was resuspended in 37% Nycodenz, overlaid with different solutions of Nycodenz, and subjected to equilibrium density gradient centrifugation. Fourteen fractions were collected from the top of the gradient, and equal volumes per fraction were analyzed by immunoblotting with antibodies against Ysl2p, Ypt51p, Vps10p, and Kex2p using ¹²⁵I-protein A as a detection system. The bands were quantified with the phosphorimager (Molecular Dynamics).

ARL1 reveals multiple genetic interactions with YSL2. (A) Δysl2 (BS747) cells were transformed with 2μm plasmids containing either ARF1, ARF2, ARL1, ARL2, or ARL3. Ura⁺ transformants were streaked out on YPD and incubated at 37°C. wt, wild type. (B) Pheromone-stimulated internalization of Ste2p was analyzed in BS694 (Δysl2) cells transformed with either 2μm-ARL1 or vector alone as described for Fig. 2A. ∗, band specifically labeled by the anti-Ste2p serum. (C) LY internalization was analyzed in BS747 (Δysl2) cells, BS747 cells transformed with CEN-based YSL2 (wild type), and BS747 cells transformed with 2μm-ARL1 as described for Fig. 2B. Cells were visualized by using fluorescence (right) and Nomarski optics (left). Bar, 10 μm. (D) Δysl2 and Δarl1 mutations are synthetic lethal. Δysl2 cells (BS695; kan^r) were crossed with Δarl1 cells (BS1103; Ura⁺). Following sporulation and tetrad dissection (47 tetrads, in total), colonies were grown at 25°C. Shown are nine tetrads (1 to 9, each A to D) that were tetratype (T) or nonparental ditype (NPD), based on auxotrophy and resistance to G-418. In all cases, the haploid progeny predicted to be G-418 resistant and Ura⁺ failed to grow at 25°C.

Δarl1 cells missort p2-CPY and are delayed in the degradation of Ste2p and α-factor. (A) BS64 (wild type [wt]) and BS1005 (Δarl1) cells were labeled for 5 min at 30°C with [³⁵S]methionine (lanes 1 and 5). Methionine was then added to initiate the chase for 5 (lanes 2 and 6), 10 (lanes 3 and 7), and 25 min (lanes 4 and 8). At each time point, aliquots of cells were removed and converted to spheroplasts after the medium (fraction E; lanes 5 to 8) was separated from the cells (fraction I; lanes 1 to 4). Fractions I and E were processed for immunoprecipitation with anti-CPY antibodies and analyzed by SDS-PAGE. p1, ER form; p2, Golgi form; m, mature form. (B) LY uptake in BS64 and BS1005 cells was monitored as described for Fig. 2B. Photographs were taken with identical exposure times by using fluorescence (left) and Nomarski optics (right). (C) Ste2p internalization assays were performed with BS64 and BS1005 cells at 30°C as described for Fig. 2A. ∗, band specifically labeled by the anti-Ste2p serum. (D) The degradation of intact and internalized ³⁵S-α-factor was quantified in strains BS64 (wild type), BS1005 (Δarl1), BS694 (Δysl2), BS1023 (ysl2-307), and BS1024 (ysl2-316) as described in Materials and Methods subsequent to internalization periods of 1, 7.5, 15, 30, and 45 min at 30°C. After an initial increase in intact, internalized α-factor the data gave a linear decay curve on a logarithmic scale. The half-lives necessary to degrade 50% of the remaining intact α-factor were 4.2 (BS64), 10.7 (BS1005), 15.7 (BS694), 15 (BS1023), and 13.2 min (BS1024).