Vps55Δ cells are defective in endocytosis of LY and FM4–64. (A) Wild-type and vps55Δ cells were incubated in the presence of 8 mg/ml LY for 30 min at 30°C as described in “Materials and Methods” and were then viewed by fluorescence microscopy using an FITC filter set. (B) Wild-type and vps55Δ cells were incubated in the presence of 10 μM FM4–64 for 30 min at 30°C as described in “Materials and Methods.” The vps27–1 cells were shifted to 37°C for 1 h and were then incubated with FM4–64. The localization of FM4–64 was detected by fluorescence microscopy using the rhodamine filter set.

Uracil permease accumulates in late endosomal/prevacuolar compartments in vps55Δ cells. (A) Wild-type and vps55Δ cells transformed with pFL38gF-GFP (expressing Fur4-GFP under the control of a galactose promoter) were grown at 30°C in galactose medium. CHX was added to exponentially growing cells. Aliquots were withdrawn at the times indicated after addition of CHX and were examined by Nomarski optics and for GFP fluorescence using the FITC filter set. (B) Wild-type and vps55Δ cells expressing Fur4-GFP and treated with CHX for 90 min were processed for immunofluorescence using the anti-Pep12p antibody followed by rhodamine-labeled goat anti-mouse IgG as described in “Materials and Methods,” except that cells were permeabilized with Triton-X100 for 10 min. The Pep12p signal was detected using the rhodamine filter set and GFP using the FITC filter set.

Vps55Δ is not affected in the retrieval of Vps10p from the prevacuolar compartment. (A) Wild-type, vps55Δ, and vps27–1 cells were shifted to 37°C for 1 h before labeling with [³⁵S]-Translabel for 15 min. The chase was performed by adding 10 mM unlabeled methionine plus cysteine. Vps10p was immunoprecipitated from the lysate at the indicated times (min) after the chase and was analyzed by fluorography after SDS-PAGE. Vps10p was clipped (denoted by an asterisk) in the vps27–1 strain. (B) The wild-type, vps55Δ, and vps27Δ strains were grown at 30°C in YPD medium. CHX was added at a final concentration of 100 μg/ml. Protein extracts were prepared at the indicated times after CHX addition and were analyzed by SDS-PAGE and immunoblotting.

Vps55p is present in the enlarged PVC of vps27–1 mutant cells. Vps27–1 cells carrying a chromosomal copy of Vps55-GFP (A) or transformed with pYEF2-GAL10-VPS55-HA (B) were grown overnight at 30°C in YPD (A) or in YNB medium supplemented with 2% lactate (B). During logarithmic growth, cells were shifted to 37°C for 3 h in YPD medium to induce the accumulation of an enlarged PVC adjacent to the vacuole. In the case of cells transformed with pYEF2-GAL10-VPS55-HA, galactose (2%) was added to induce Vps55p synthesis when cells were shifted at a restrictive temperature. Vps27–1 cells producing the Vps55-GFP fusion protein were viewed directly using an FITC filter set. Vps27–1 cells producing the Vps55-HA fusion protein were fixed and processed for immunofluorescence using the monoclonal anti-HA antibody followed by rhodamine-labeled goat anti-mouse IgG. Fluorescence was viewed using rhodamine filter set, and cell morphology was observed with Nomarski optics. Note the altered cellular morphology in cells expressing the Vps55-HA fusion protein compared with living cells producing the Vps55-GFP fusion protein.

Subcellular fractionation of cells expressing tagged Vps55p. Wild-type and vps35Δ cells expressing chromosomal integrated Vps55-HA were lysed with glass beads and extracts were fractionated by differential centrifugation. The proteins were separated by SDS-PAGE and were analyzed by immunoblotting for the presence of Vps55-HA and various marker proteins (an asterisk indicates clipped Vps10p; ALPm, ALP mature; ALPs, ALP soluble).

Human OB-RGRP complements the disruption of VPS55. (A) Wild-type and vps55Δ strains transformed with pYEF2, pYEF2-GAL10-VPS55-HA, or pYEF2-GAL10-OB-RGRP-HA were cultured overnight in YNB medium supplemented with 2% glucose. Exponentially growing cells (10⁵ cells/spot) were plated on YNB supplemented with 2% galactose to induce the production of Vps55p and OB-RGRP, and were cultured for 48 h in contact with a nitrocellulose membrane. Wild-type and vps55Δ cells transformed with the centromeric pYCG-VPS55 were treated in the same way. Secreted CPY bound on the nitrocellulose membrane was probed using a monoclonal antibody raised against CPY. (B) The strains listed above were transformed with pgF (expressing uracil permease under the control of a galactose promoter) and were grown overnight in YNB medium supplemented with 2% lactate. Galactose (2%) was added to the cells for 3 h during exponential growth to induce uracil permease, Vps55p, and OB-RGRP synthesis. Protein extracts were prepared at the indicated times (min) after CHX addition and were analyzed by SDS-PAGE and immunoblotting. Uracil permease (Fur4p) was detected using a polyclonal antibody.

CPY maturation is delayed and p2-CPY is secreted in the medium in vps55Δ cells. (A) Total protein extracts were prepared from exponentially growing wild-type, vps55Δ, sec7–1, apm3Δ, and pep4 cells. ALP and CPY were analyzed by SDS-PAGE and immunoblotting. The positions of the precursors (p2-CPY and proALP) and the mature (m) forms are indicated. In addition to proALP, all cells also display a lumenal aberrantly processed soluble form of ALP, as described by Stepp et al. (1997). (B) vps55Δ, vps1, and end13–1/vps4 cells were grown on YPD plates (10⁵ cells/spot) for 48 h in contact with a nitrocellulose membrane. Secreted CPY was detected on the membrane by probing with a mAb directed against CPY. (C) Wild-type, vps55Δ, and vps1Δ cells were labeled with [³⁵S]-Translabel for 10 min and chased for the indicated times in the presence of 10 mM unlabeled methionine plus cysteine. CPY was precipitated either from total cell culture (0, 5, 10, 20, and 40 min) or after the separation of cells (I, intracellular) and medium (E, extracellular; 40 min of chase). Aliquots corresponding respectively to 1 vol (total cell culture), 3 vol (I), or 6 vol (E) of original proteins were analyzed by SDS-PAGE, and radioactivity was detected by fluorography. As no p2-CPY was recovered in the intracellular fractions after 40 min of chase, the percentage of CPY secretion was roughly estimated from the overall CPY pattern in total cell culture at 40 min. The recovery of immunoprecipitated proteins was lower for I and E than in T. The ER (p1), Golgi (p2), and mature (m) forms of CPY are indicated.

Vps55Δ cells are impaired in the uracil permease endocytic pathway. (A) Wild-type and vps55Δ cells transformed with pFL38gF (producing Fur4p under the control of a galactose promoter) were grown to the exponential growth phase in galactose medium. CHX was added to a final concentration of 100 μg/ml, and uracil uptake was measured at the indicated times. Results are presented as a percentage of initial activity. (B) Aliquots were collected at the indicated times after CHX addition and protein extracts were prepared. Proteins from 1.5 × 10⁷ cells were analyzed for uracil permease (Fur4p) by immunoblot.

Vps55Δ cells do not accumulate a class E compartment. The distribution of Pep12p and Vat2p in wild-type, vps55Δ, and vps27–1 cells was determined by immunofluorescence. Exponentially growing cells were fixed and permeabilized with 1% Triton-X100 for 5 min to preserve vacuolar morphology. Immunofluorescence was performed with monoclonal anti-Pep12p or anti-Vat2p antibodies. Fluorescence was viewed using rhodamine filter set and cell morphology was observed with Nomarski optics.

Vps55p is a stable integral membrane protein localized in small punctuate structures. (A) Cells expressing Vps55-HA were grown to exponential phase, converted to spheroplasts, and lysed with glass beads. Cell lysates were treated with lysis buffer alone (control), 1% Triton-X100, or 0.1 M sodium carbonate (pH 11). The initial homogenate (H) was separated into supernatant (S) and pellet (P) by centrifugation at 100,000 × g. Samples were analyzed by SDS-PAGE and immunoblotting using a monoclonal anti-HA antibody. (B) Wild-type strain expressing Vps55-HA was labeled with [³⁵S]-Translabel for 15 min and was chased for the indicated times in the presence of 10 mM unlabeled methionine plus cysteine. Vps55-HA was immunoprecipitated from the cell lysate at the indicated times using a monoclonal antibody directed against the HA tag. Vps55-HA was analyzed by fluorography after SDS-PAGE. (C) Cells producing the Vps55-GFP fusion protein were viewed with a microscope (Leica) using the FITC filter set and Nomarski optics. An image of a budding cell at higher magnification is presented.

Vps55p partially colocalizes with Sec7-GFP. Wild-type cells harboring chromosomal copies of SEC7-GFP and VPS55-MYC were grown overnight in YPD medium. Cells were fixed and processed for immunofluorescence using the monoclonal anti-Myc antibody followed by rhodamine-labeled goat anti-mouse IgG. GFP fluorescence was visualized with an FITC filter set, and HA distribution was observed with a rhodamine filter set. Arrows indicate Myc staining colocalizing with Sec7p-GFP.

Human OB-RGRP displays the same distribution as Vps55p. Vps27–1 and wild-type cells producing the chromosomal encoded Sec7-GFP were transformed with pYEF2-GAL10-OB-RGRP-HA. Cells were cultured overnight in YNB medium supplemented with 2% lactate. Galactose (2%) was added and the cells were shifted to 37°C for 3 h. Cells were fixed and processed for immunofluorescence using the monoclonal anti-HA antibody followed by rhodamine-labeled goat anti-mouse IgG. The FITC filter set was used to visualize GFP fluorescence, and the rhodamine filter set was used to visualize HA fluorescence. Arrows indicate colocalized Vps55-HA and Sec7-GFP, and arrowheads indicate HA staining not colocalized with Sec7p-GFP.