PMC

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) ().

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.

Secretion of active α-factor from various deletion strains. MATα 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.

Secretion of highly glycosylated α-factor precursor in ypt6/sys3 double deletion mutants. MATα 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.

Kex2p steady-state levels in ypt6/sys3 null mutants. (A) Cell extracts were prepared from cultures of wild-type (wt), Δypt6, Δsys3, and Δypt6/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 Δkex2 strain was included in the analysis.

FM4-64 staining of vacuolar membranes in ypt6/sys3 null mutants. Wild-type, Δsys3, Δypt6, and Δypt6/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.

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 ().

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.

Synthetic negative growth phenotype of double deletion mutants. Growth curves of wild-type (▾), Δypt6 (□), Δsys3 (♦), and Δypt6/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.

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.

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.

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.