FM4-64 recycling is inhibited in rcy1Δ, tlg1Δ, and tlg2Δ cells. All FM4-64 recycling measurements were done as described in Fig. 7. (A and B) Recycling of FM4-64 at 24°C is strongly inhibited in rcy1Δ (A, grey line), tlg1Δ (B, grey line), and tlg2Δ (B, dark grey line) compared with their corresponding wild-type (black lines) cells. vps4 (C, grey line) and ypt7 (D, grey line) recycle FM4-64 at similar rates as the corresponding wild-type cells (black lines).

Accumulation of LY in punctuate nonvacuolar structures close to and in the bud of the rcy1-null mutant cells. rcy1 mutant (A and C) and wild-type cells (B) were incubated in YPUAD containing LY (4 mg/ml) for 1 h at 24°C. The cells were then washed and visualized using fluorescence optics. (C) rcy1 mutant cells visualized at higher magnification using either fluorescence (left panels) or Nomarski optics (right panels).

Tlg1p localizes to the enlarged compartment of the rcy1Δ mutant. Logarithmically growing cells were fixed, dehydrated, and embedded for EM as described in Materials and Methods. Cell sections were then incubated with purified anti-Tlg1p antibodies followed by anti-IgG secondary antibodies. The gold-coupled secondary antibodies can be seen on the sections as black dots with a diameter of 10 nm. Immunogold-labeled sections of rcy1Δ (A–C) and the corresponding wild-type (D–F) are shown. Bars, 200 nm. (D) Tlg1p-positive membranes are indicated by a star.

Tlg1p localizes to the enlarged compartment of the rcy1Δ mutant. Logarithmically growing cells were fixed, dehydrated, and embedded for EM as described in Materials and Methods. Cell sections were then incubated with purified anti-Tlg1p antibodies followed by anti-IgG secondary antibodies. The gold-coupled secondary antibodies can be seen on the sections as black dots with a diameter of 10 nm. Immunogold-labeled sections of rcy1Δ (A–C) and the corresponding wild-type (D–F) are shown. Bars, 200 nm. (D) Tlg1p-positive membranes are indicated by a star.

Degradation of α-factor pheromone and the plasma membrane protein uracil permease is defective in the rcy1Δ mutant cells. Internalization (A and B) and degradation (C–F) of α-factor (A, C, and E) and uracil permease (B, D, and F) was studied. (A) Internalization of α-factor was followed as the ratio of internalized radio-labeled α-factor (resistant to pH 1.1 wash) divided by the total cell-associated cpm (internalized plus surface receptor-bound α-factor retained after pH 6 wash). rcy1Δ cells (solid line) internalized [^S35]α-factor at rates similar to wild-type cells (dashed line). (B) Transport of [³H]uracil into yeast cells was used as a measure for the relative amounts of uracil permease exposed at the cell surface. Upon addition of cycloheximide (100 μg/ml), uracil permease levels at the cell surface decreased. Uracil uptake is plotted as the percentage activity at the initial timepoint. The decrease of uracil transport over time is identical in the rcy1Δ (solid line) and wild-type cells (dashed line). (C) Degradation of α-factor. Cells were allowed to take up radiolabeled α-factor as described in A. Membrane traffic was stopped and cells were washed in cold buffer, pH 1.1, at the indicated timepoints in minutes. Cells were extracted and cell-associated radioactivity was analyzed by TLC and autoradiography. The disappearance of intact (i) α-factor was quantified (E). In wild-type cells, degraded α-factor (d) was already observed 20 min after internalization at 24°C had been initiated. (D) Degradation of uracil permease. At the indicated times after addition of cycloheximide (100 μg/ml), total proteins were extracted. The intensity of the uracil permease band as detected on the Western blot was quantified and the values presented as the relative amounts of the signal before adding cycloheximide (F). (E and F) Wild-type (dashed line) and rcy1Δ (solid line).

Ste2p traffic is blocked in an endocytic compartment. (A) Ligand-induced endocytosis of Ste2p was followed over time. rcy1Δ (left column) and wild-type cells (right column) were exposed to α-factor (10⁻⁷ M) for the indicated times in minutes. Cells were fixed and stained using a polyclonal antibody against Ste2p. Note the large Ste2p-positive membranes in the rcy1Δ strain. (B) For comparison of rcy1Δ (left) and wild-type strains (right) the cells were quantified for the presence of Ste2p-positive compartments. The sizes of Ste2p-positive compartments were judged by immunofluorescence (not real size of compartments). The percentage of total cells with Ste2p-positive structures with an apparent size >1 μm at its largest diameter is represented by the black bars. The grey bars show the percentage of cells with smaller round Ste2p-positive compartments of ∼0.5–1 μm in diameter. (C) Pma1p does not colocalize to the internal Ste2p-positive membranes. A wild-type strain (right panels) and the rcy1Δ mutant strain (left panels) expressing a genomic hemagglutinin-tagged version of Pma1p were labeled for Ste2p as in A (top panels) and Pma1HA using a monoclonal anti-hemagglutinin (bottom panels) by double immunofluorescence. No cross-bleeding was observed when either of the two primary antibodies was omitted (not shown).

CPY targeting and invertase secretion are normal in the rcy1Δ mutant. (A) Maturation of CPY occurs at a normal rate in rcy1Δ mutant cells. Wild-type (left lanes) and rcy1Δ (right lanes) cells were pulsed for 4 min with 150 μCi/ml of [³⁵S]methionine/cysteine labeling mixture and chased for the indicated times (see Materials and Methods). CPY was immunoprecipitated from total protein extracts. The ER form of CPY (p1CPY) and the Golgi form of CPY (p2CPY) disappear in both strains at approximately equal rates. (B) Like wild-type cells (WT), rcy1Δ cells do not secrete CPY. Secretion of CPY was assayed on nitrocellulose filters. vps mutants secrete a fraction of CPY, which binds to nitrocellulose filters and can be detected by probing with a CPY-specific antibody. Due to inefficient sorting of CPY in the vps mutant (vps1) and the class E vps mutant (vps4), a fraction of CPY is secreted, which results in a strong signal on the membrane. (C) Invertase secretion occurs at normal rates from rcy1Δ cells. Cells grown in YPUAD (2% glucose) were washed and resuspended in a medium containing 2% sucrose and 0.05% glucose at time 0. Samples were taken at different timepoints after this induction and assayed for secreted invertase activity (see Materials and Methods). Both the onset and the extent of invertase secretion from rcy1Δ cells (solid line) and wild-type cells (dashed line) were similar.

Differences of FM4-64 traffic in rcy1Δ (left) and wild-type (right) strains. (A) Cells were incubated for 10 min in YPUAD in the presence of 20 μM FM4-64. The cells were then washed in fresh medium to remove the surface-bound dye and further incubated. Samples were taken and stopped in cold buffer containing 10 mM NaN₃ and 10 mM NaF. The times on the left of the figure indicate the minutes after addition of the dye. The cells were visualized using fluorescence optics. (B) For comparison of vacuolar FM4-64 staining (top), Nomarski images (bottom) are shown for the 20-min timepoint.

Measuring recycling in yeast. Cells were allowed to take up FM4-64 (40 μM) for 12 min as described in Fig. 6. The cells were then washed in ice-cold SD minimal medium. Thoroughly washed cells were resuspended in SD medium at 24°C. FM4-64 fluorescence in the cell suspension was measured over time in a spectrofluorometer (see Materials and Methods). (A) Fluorescence associated with wild-type cells (black line) decreases over time. This decrease was blocked when the cells were resuspended in SD medium containing NaN₃ and NaF (grey line).

FM4-64 is recycled into the medium in a SEC18-dependent manner. (A) Wild-type cells (grey bars) or rcy1Δ cells (black bars) were loaded with FM4-64 at 24°C, washed, and incubated for the indicated time. Relative fluorescence changes seen after addition of 1% CHAPS were quantified. The fluorescence change seen after 8 min with wild-type cells was set to 100%. (B) Aliquots of recycling cells prepared as in A were treated with (+) or without (−) 1% CHAPS for 1 min. Then, 10 mM sodium azide and 10 mM sodium fluoride were added. After centrifugation, the fluorescence inside the cells was measured and divided by the cell number. After resuspension of cells that did not receive CHAPS (−) in fresh medium, 1% CHAPS was added (−/+). No increase in total fluorescence was observed upon addition of CHAPS under these conditions. In agreement with continuous measurements (Fig. 7), the cell pellet analyzed at 2 min contained about twice as much fluorescence as the cells that had recycled for 8 min. The fluorescence in the pellet of wild-type cells at 8 min was set to 100%. (C and D) Wild-type (black lines) and sec18-1 (grey lines) mutant cells were resuspended in SD medium prewarmed to 23°C (B) or 36°C (C). At 36°C, the recycling of FM4-64 was strongly inhibited in the sec18-1 strain compared with the wild-type. (E) Quantitation of the fluorescence changes in response to 1% CHAPS of the different mutants used in this study. The average of two independent experiments is shown for each mutant and condition. The fluorescence change due to addition of CHAPS seen after 8 min with wild-type cells was set to 100%. Standard error bars are indicated where more than two independent experiments have been performed.

Model for membrane traffic in the vacuolar system. (1) Traffic from the postulated sorting endosome to the PVC is inhibited in the rcy1Δ mutant. (2) Two possible pathways that contribute to recycling from the sorting endosome to the plasma membrane, one passing through the Golgi and the other directly to the plasma membrane. (3) Traffic steps blocked in class E vps mutants, which do not affect recycling. (4) Traffic from the late endosome to the vacuole, which is also not required for recycling.