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. 2007 Apr;18(4):1203-19.
doi: 10.1091/mbc.e06-11-1035. Epub 2007 Jan 17.

Avl9p, a member of a novel protein superfamily, functions in the late secretory pathway

Edina Harsay  1 , Randy Schekman
Affiliations

Avl9p, a member of a novel protein superfamily, functions in the late secretory pathway

Edina Harsay et al. Mol Biol Cell. 2007 Apr.

Abstract

The branching of exocytic transport routes in both yeast and mammalian cells has complicated studies of the late secretory pathway, and the mechanisms involved in exocytic cargo sorting and exit from the Golgi and endosomes are not well understood. Because cargo can be sorted away from a blocked route and secreted by an alternate route, mutants defective in only one route do not exhibit a strong secretory phenotype and are therefore difficult to isolate. In a genetic screen designed to isolate such mutants, we identified a novel conserved protein, Avl9p, the absence of which conferred lethality in a vps1Delta apl2Delta strain background (lacking a dynamin and an adaptor-protein complex 1 subunit). Depletion of Avl9p in this strain resulted in secretory defects as well as accumulation of Golgi-like membranes. The triple mutant also had a depolarized actin cytoskeleton and defects in polarized secretion. Overexpression of Avl9p in wild-type cells resulted in vesicle accumulation and a post-Golgi defect in secretion. Phylogenetic analysis indicated evolutionary relationships between Avl9p and regulators of membrane traffic and actin function.

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Figures

Figure 1.
Figure 1.
The vps1Δ and apl2Δ mutations have unique effects on invertase sorting into vesicles accumulated in a sec6-4 mutant background. We used a Nycodenz density gradient fractionation assay in which secretory vesicles accumulated by a sec6-4 mutant reproducibly peak in fraction 8 or 9 (light-density vesicles) or fractions 16–18 (high-density vesicles). (A) Nycodenz gradient fractionation of secretory vesicles accumulated in a sec6-4 vps1Δ mutant. All cargo is sorted into light-density vesicles, indicating a defect in the dense-vesicle transport pathway, as described previously (Harsay and Schekman, 2002). (B) The apl2Δ sec6-4 mutant accumulates cargo primarily at a density intermediate between that of vesicles in the light-vesicle and dense-vesicle pathways. (C) The gga1Δ gga2Δ mutations have a relatively small effect on exocytic cargo transport in a sec6-4 strain background. Invertase, which is a dense-vesicle cargo, is shown. The gradient density profile shown was highly reproducible for all gradients.
Figure 2.
Figure 2.
The avl9-1 mutant is synthetically lethal in an apl2Δ vps1Δ strain background. The lethality of an avl9-1 apl2Δ vps1Δ strain carrying a URA3-APL2 plasmid (or URA3-VPS1 plasmid, not shown) on 5-FOA is rescued by introducing a TRP1 plasmid containing either the VPS1 or APL2 genes.
Figure 3.
Figure 3.
The avl9Δ apl2Δ vps1Δ mutant has defects in the exocytic pathway. (A) Western blot showing the accumulation of internal Bgl2p. The avl9Δ apl2Δ vps1Δ strain carrying a plasmid with AVL9 under the control of the GAL1 promoter was grown in medium containing 2% galactose, 0.3% glucose for optimal growth and then shifted to medium with 2% glucose for 20 h to deplete Avl9p. Bgl2p is primarily in the cell wall at steady state, so internal accumulation can be detected by removing the cell wall. PGK is a cytoplasmic protein used as loading control. (B) Metabolic labeling and pulse-chase analysis shows a kinetic defect in Bgl2p transport that becomes more pronounced with increased time in glucose to deplete Avl9p. Cells were pulse-labeled for 5 min, and chase was 60 min in each experiment. Cell walls were removed, and cells (I, inside) were separated from the cell wall and media fraction (O, outside). Bgl2p was immunoprecipitated and detected by SDS-PAGE and phosphorimaging. (C) Invertase secretion was assayed as described in Materials and Methods. External and total invertase levels were determined by a colorimetric enzyme assay to calculate percent secretion. The means of three experiments for each strain are shown. Error bars, SEM.
Figure 4.
Figure 4.
The avl9Δ apl2Δ vps1Δ mutant accumulates Golgi-like structures. (A–D) An avl9Δ apl2Δ vps1Δ strain carrying a plasmid with AVL9 under the control of the GAL1 promoter was grown as in Figure 3 to deplete Avl9p. Cells were prepared for thin-section EM after a 20-h shift to glucose. (E) An avl9Δ strain, prepared as above. (F) The avl9Δ apl2Δ vps1Δ mutant carrying a plasmid with AVL9 under the control of its native promoter, prepared as above. Bars, 500 nm.
Figure 5.
Figure 5.
The avl9Δ sec6-4 mutant accumulates cargo primarily in intermediate-density membranes, similar to what was observed for apl2Δ sec6-4. Density gradient fractionation was performed as for Figure 1.
Figure 6.
Figure 6.
The avl9Δ mutation perturbs vesicle formation in a sec6-4 strain background at restrictive temperature. Cells were grown to log-phase at 24°C and shifted to 36°C for 45 min before fixing for thin-section EM. (A–C) avl9Δ sec6-4 mutant accumulates Golgi-like membranes and vesicles that are smaller than vesicles accumulated in a sec6-4 mutant (D). Bars, 500 nm.
Figure 7.
Figure 7.
Polarized secretion is defective in avl9 mutants. Cells were stained with Alexafluor-568 phalloidin to label polymerized actin (A–D) or with calcofluor to label chitin (E–I). (A) wt; (B) apl2Δ vps1Δ: (C) avl9Δ diploid; (D) avl9Δ apl2Δ vps1Δ after depleting Avl9p, as described in Materials and Methods; (E) apl2 vps1; (F, G) avl9Δ apl2Δ vps1Δ after depleting Avl9p; (H) wt diploid; (I) avl9 diploid. All cells are at the same magnification except the DIC (differential interference contrast microscopy) inset in D. All cells are haploid unless otherwise noted. Arrows indicate abnormal budding pattern in haploid cells.
Figure 8.
Figure 8.
The overexpression of Avl9p is toxic. Strains EHY1252 (carrying a URA3 CEN plasmid vector backbone) and EHY1253 (containing a URA3 CEN plasmid with GAL1p::AVL9) were streaked on CSM, −Ura plates with either 2% galactose or 2% glucose and grown for 3 days (galactose) or 2 days (glucose).
Figure 9.
Figure 9.
The overexpression of Avl9p results in perturbation of membrane organelles. The strains described in Figure 8 were grown as described in Materials and Methods and processed for thin-section EM soon after growth rate slowed down in galactose medium. (A) Wild-type cells grown under these conditions have a dense cytoplasm with small punctate structures. (B) Cells overexpressing Avl9p accumulate heterogeneous vesicles and expanded ER membranes. The images are at the same magnification. Bar, 500 nm.
Figure 10.
Figure 10.
Overexpression of Avl9p results in a post-Golgi secretory defect. (A) The strains described in Figure 8 were grown as described in Materials and Methods, and growth after shift to galactose-containing medium was monitored. A slow-down in growth-rate was detectable by 9 h in galactose for cells with GAL1p::AVL9. (B) Comparison of the hourly growth rate (fold increase in OD600 after 1 h of growth) for cells containing a URA3 CEN plasmid with GAL1p:: AVL9 or an empty URA3 CEN vector. For 3–9 h, n = 6; for 9–15 h, n = 4. Error bars, SEM. (C) Internal Bgl2p was assayed at the indicated times after shift to galactose, as described for Figure 3. (D) Pulse-chase analysis of CPY transport after 14 h in galactose-containing medium, performed as described in Materials and Methods.
Figure 11.
Figure 11.
Conserved regions between Avl9 superfamily members, designated AH1-AH5, have similar secondary structure predictions. Predicted alpha helices are red and beta folds are blue. All proteins are drawn at the same scale, which is indicated in amino acid residues. Full-length proteins are shown. For DENNd1c, predicted structures are shown only for the uDENN, DENN, and dDENN regions. In some proteins, including MesA, AH5 is interrupted by an unconserved region.
Figure 12.
Figure 12.
An inferred phylogenetic tree showing the relationships among Avl9 superfamily proteins and DENN domains, based on aligned AH1-4 and uDENN/DENN regions. Branch lengths were estimated by the maximum likelihood program PROML from the Phylip software package. Bootstrap values are indicated for both distance and parsimony (parenthesis) methods. Only one number is given for branching that differed between the two methods. The accession numbers for the sequences used are listed in Materials and Methods. The alignment used to produce the tree is shown in Supplementary Figure 4B.
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