Structural and functional analysis of a novel coiled-coil protein involved in Ypt6 GTPase-regulated protein transport in yeast - PubMed (original) (raw)

Structural and functional analysis of a novel coiled-coil protein involved in Ypt6 GTPase-regulated protein transport in yeast

M Tsukada et al. Mol Biol Cell. 1999 Jan.

Free PMC article

Abstract

The yeast transport GTPase Ypt6p is dispensable for cell growth and secretion, but its lack results in temperature sensitivity and missorting of vacuolar carboxypeptidase Y. We previously identified four yeast genes (SYS1, 2, 3, and 5) that on high expression suppressed these phenotypic alterations. SYS3 encodes a 105-kDa protein with a predicted high alpha-helical content. It is related to a variety of mammalian Golgi-associated proteins and to the yeast Uso1p, an essential protein involved in docking of endoplasmic reticulum-derived vesicles to the cis-Golgi. Like Uso1p, Sys3p is predominatly cytosolic. According to gel chromatographic, two-hybrid, and chemical cross-linking analyses, Sys3p forms dimers and larger protein complexes. Its loss of function results in partial missorting of carboxypeptidase Y. Double disruptions of SYS3 and YPT6 lead to a significant growth inhibition of the mutant cells, to a massive accumulation of 40- to 50-nm vesicles, to an aggravation of vacuolar protein missorting, and to a defect in alpha-pheromone processing apparently attributable to a perturbation of protease Kex2p cycling between the Golgi and a post-Golgi compartment. The results of this study suggest that Sys3p, like Ypt6p, acts in vesicular transport (presumably at a vesicle-docking stage) between an endosomal compartment and the most distal Golgi compartment.

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Figures

Figure 1

Figure 1

Comparison of Sys3p and Uso1p amino acid sequences. Identical residues are on a black background, and similar residues are shaded. The alignment was made using the program PILEUP (Genetics Computer Group, Inc., Madison, WI) (Feng and Doolittle, 1987).

Figure 2

Figure 2

Synthetic negative growth phenotype of double deletion mutants. Growth curves of wild-type (▾), Δ_ypt6_ (□), Δ_sys3_ (♦), and Δ_ypt6/sys3_ (×) strains are shown. Precultures of corresponding strains were grown to stationary phase at 25°C. Cells were diluted into fresh YEPD medium and incubated overnight to an OD600 of 1–3. Cells were again diluted into fresh YEPD medium to an OD600 of ∼0.05 and incubated at 30°C. Cell growth was followed by measuring the optical density at 600 nm.

Figure 3

Figure 3

Sorting of the vacuolar enzyme CPY. Wild-type (wt) cells and strains carrying gene deletions as indicated were grown to exponential phase, spheroplasted, labeled for 15 min at 25°C with Tran35S-label, and chased for 30 min at 25°C. The labeled spheroplasts were separated into pellet (intracellular [I]) and supernatant (extracellular [E]) fractions. The presence of CPY in these fractions was determined by SDS-PAGE of these proteins immunoprecipitated with anti-CPY antibodies.

Figure 4

Figure 4

Secretion of active α-factor from various deletion strains. _MAT_α strains carrying gene deletions as indicated were grown to stationary phase, and after appropriate dilution, 2.5-μl cultures were spotted onto a lawn of the MATa supersensitive strain. The diameter of the growth-inhibitory zone (halo) is proportional to the amount of α-factor secreted.

Figure 5

Figure 5

Secretion of highly glycosylated α-factor precursor in ypt6/sys3 double deletion mutants. _MAT_α strains carrying gene deletions as indicated were grown to exponential phase and labeled for 30 min at 25°C with Tran35S-label. Culture media were collected, and α-factor was immunoprecipitated with anti-α-factor antiserum and analyzed by SDS-PAGE on 15% gels. α (p), highly glycosylated α-factor precursor; α (m), mature α-factor.

Figure 6

Figure 6

Kex2p steady-state levels in ypt6/sys3 null mutants. (A) Cell extracts were prepared from cultures of wild-type (wt), Δ_ypt6_, Δ_sys3_, and Δ_ypt6/sys3_ strains, and levels of Kex2p were analyzed by SDS-PAGE (6% polyacrylamide) and immunoblotting using a polyclonal antiKex2p antibody. (*) Position of a possible Kex2p degradation product. (B) Analysis of Kex2p levels were determined as in A, except that cell extracts were prepared from cultures of pra1-1/prb1-1/prc1-1 versions of each strain, and a Δ_kex2_ strain was included in the analysis.

Figure 7

Figure 7

Morphological alterations in ypt6/sys3 deletion mutant cells. Logarithmically growing cells were fixed with potassium permanganate to highlight membrane structures. Neighboring cells of one section are shown to document the specificity of the alterations. (A) Single sys3 disruptants shown here and wild-type cells were indistinguishable. (B) Cells of a ypt6/sys3 null mutant strain. Arrowheads point to clearly identifiable vacuoles; arrows point to the spherical structures. (C) Higher magnification of a double mutant cell showing the accumulation of 40- to 50-nm vesicles. V, vacuole; E, endoplasmic reticulum; N, nucleus; M, mitochondria. Bars, 1 μm.

Figure 8

Figure 8

FM4-64 staining of vacuolar membranes in ypt6/sys3 null mutants. Wild-type, Δ_sys3_, Δ_ypt6_, and Δ_ypt6/sys3_ cells were grown at 25°C, labeled for 1 h with 30 μM FM4-64, washed three times with cold PBS buffer, and chased in YPD for 2 h. Then cells were examined by Nomarski and fluorescence microscopy for FM4-64 fluorescence. Essentially the same result was obtained after a 30-min chase time.

Figure 9

Figure 9

Subcellular localization of the Sys3 protein. Yeast cells lacking vacuolar proteinases (strain cl3-ABYS-86) were grown to exponential phase at 30°C in YEPD medium and disrupted with glass beads. Unbroken cells were removed by centrifugation at 500 × g. The soluble fraction (S1) was separated into pellet (P2) and supernatant (S2) fractions by centrifugation for 10 min at 10,000 × g. The S2 fraction was then centrifuged for 1 h at 100,000 × g to generate soluble (S3) and pellet (P3) fractions. Equal portions of the different fractions were analyzed by SDS-PAGE (6% polyacrylamide) and immunoblotting using anti-Sys3p antibodies.

Figure 10

Figure 10

Gel filtration of Sys3 protein. Soluble proteins (S3 fraction prepared in the presence of 0.5 M KCl) were chromatographed on Sephacryl 400. The absorbtion profile of the eluted fractions is shown. Arrowheads (from left to right) show the positions of marker proteins (thyroglobulin [670 kDa], ferritin [440 kDa], catalase [232 kDa], γ-globulin [158 kDa], ovalbumin [44 kDa], and myoglobin [17 kDa]) and dextran 2000. Eluted fractions were analyzed for Sys3p and Sys2p by SDS-PAGE and Western blotting. The positions of molecular size standards used to calibrate the column are shown below the fraction numbers.

Figure 11

Figure 11

Oligomerization of Sys3p. A cytosolic S3 fraction (9 mg/ml) was incubated with the cross-linker BS3 at the concentrations indicated. After SDS-PAGE, immunoblot analysis was performed using anti-Sys3p antibodies. The positions of molecular mass markers are given to the left.

Figure 12

Figure 12

Sys3 protein interactions in the two-hybrid system. The Y190 reporter strain was transformed with either pASI-SYS3, pASI-YPT6(Q69L), or pASI-YPT6 (wild-type) as baits in combination with either pACTII-SYS3 or pACTII-GYP6 as prey. β-Galactosidase activity was detected by the 5-bromo-4-chloro-3-indolyl galactopyranoside filter assay. The lack of transcription activation by the baits alone is shown for Sys3p and Ypt6(Q69L)p. The functionality of the Gal4–Ypt6 fusion proteins is confirmed by their strong interaction with the GTPase-activating protein Gyp6p (Strom et al., 1993).

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