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First published online 1 June 2004
doi: 10.1242/jcs.01157

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A role for yeast oxysterol-binding protein homologs in endocytosis and in the maintenance of intracellular sterol-lipid distribution

Christopher T. Beh^*, and Jasper Rine

Department of Molecular and Cell Biology, University of California, 401 Barker Hall, Berkeley, CA 94720, USA

View larger version (94K): [in a new window]   Fig. 1. Electron micrographs of Osh protein-depleted cells and OSH temperature-sensitive mutants. (A) Wild-type, (B) osh PMET3-OSH2, and (C and D) osh osh4(ts) cells, were examined by electron microscopy. V, vacuole; N, nucleus; LD, lipid droplets. Thick arrows indicate the large dark irregular structures that correspond to fragmented and collapsed vacuoles, thin arrows indicate vesicles, asterisks indicate cell wall abnormalities. (A) Wild-type cells (SEY6210) contained an average of 2.5 lipid droplets (s.d.=2.1; 54 random sections counted); only 2% of wild-type cells (1/54) accumulated more than twenty-five 50 nm vesicles; 2% of wild-type cells (1/54) had pronounced cell wall defects; 2% exhibited vacuolar fragmentation (1/54); and in only 10% of sections with vacuoles (5/50) were <100 nm vacuolar droplets observed. (B) In the absence of methionine (-Met), OSH2 was expressed in osh PMET3-OSH2 cells (JRY6326), which were viable, but compared with wild-type cells defects were nonetheless observed. In random sections, an average of 2.6 lipid droplets were counted (s.d.=2.5; 70 sections counted); 6% of growing osh PMET3-OSH2 cells (4/63) exhibited cell wall defects; 5% had an accumulation of more than 25 50 nm vesicles (4/75); 39% exhibited vacuolar fragmentation (25/64); and in 61% of vacuole-containing sections (37/61), small <100 nm vacuolar lipid droplets were observed. After methionine addition (+Met) and the gradual depletion of Osh proteins in osh PMET3-OSH2 cells, the average number of lipid droplets was 8.1 (s.d.=6.7; 30 sections counted); 47% of Osh-depleted cells (14/30) had pronounced cell wall defects; in 30% of random sections (9/30) more than twenty-five 50 nm vesicles accumulated; 80% of random sections (24/30) contained aberrant, dark-staining vacuolar remnants; and in 81% of sections with vacuoles (26/32), <100 nm vacuolar lipid droplets were observed. (C) osh osh4-1 cells, and (D) osh osh4-2 cells were cultured either at 23°C or at 23°C and then for 1 hour at 37°C. For osh osh4-2 cells at 23°C, an average of 3.5 lipid droplets were observed (s.d.=3.4; 41 sections counted); 37% exhibited vacuolar fragmentation (18/49); 9% accumulated more than twenty-five 50 nm vesicles (4/46); and 10% had pronounced cell wall defects (6/62). At 37°C, osh osh4-2 cells contained an average of 4.4 lipid droplets (s.d.=4.8; 52 sections counted); 62% exhibited vacuolar fragmentation (29/47); 10% accumulated more than twenty-five 50 nm vesicles (6/61); and 15% had pronounced cell wall defects (10/65). Bars, 1 µm.

View larger version (46K): [in a new window]   Fig. 2. Osh protein depletion caused defective chitin accumulation. Calcofluor staining of yeast cells viewed by fluorescence microscopy shows chitin deposition. (A) Chitin bud scars are visible in wild-type cells (SEY6210) at the mother-bud junction or immediately adjacent, which reflects the axial pattern of haploid budding. (B) In osh PMET3-OSH2 cells (JRY6326) after methionine repression of OSH2 expression, Osh depletion caused chitin to accumulate at abnormal sites other than just the mother-bud junction (e.g. bud tip). Arrows indicate abnormal deposits of chitin. Exposure times for osh PMET3-OSH2 cells were shorter that wild type indicating a comparatively greater amount of chitin.

View larger version (59K): [in a new window]   Fig. 3. CPY maturation and sorting was unaffected by loss of all OSH function. (A) Autoradiograph of CPY immunoprecipitations from wild-type (Osh+; SEY6210), osh osh4-1 (CBY886) and osh OSH4 (JRY6320) cultures. p1, ER/early Golgi form of CPY; p2, late Golgi CPY; m, mature vacuolar form of CPY. Cells were pulse-labeled with Tran[35S]-label for 5 minutes and chased for the periods indicated at 37°C. CPY was immunoprecipitated from cell lysates and analyzed by SDS-PAGE. (B) CPY immunoblots of vps4 (CBY824), wild-type (W.T.; SEY6210), and osh PMET3-OSH2 (JRY6326) strains. Strains were spotted on solid media with increasing concentrations of methionine (up to 100 mM), which repressed expression of OSH2 in the osh PMET3-OSH2 strain and arrested growth. Filters were placed directly on the solid medium and CPY secreted from cells was detected with anti-CPY antibodies (Roberts et al., 1991). Filters were also probed with anti-Act1p to detect the discharge of actin from non-specific cell lysis. Equivalent amounts of cells were spotted on the solid medium as shown by the example in the bottom panel.

View larger version (46K): [in a new window]   Fig. 4. Secretion of Hsp150p to the cell surface was unaffected by OSH temperature-sensitive mutants. Autoradiograph of Hsp150 purified using Con A-affinity chromatography from the extracellular medium and cell extracts of wild-type (Osh+; SEY6210), osh osh4-1 (CBY926), osh OSH4 (CBY924), wild-type (W.T.; RSY255) and sec1-1 (RSY782) strains. Cultures were incubated at 37°C for 60 minutes and, after a 10 minute pulse with [35S]-label followed by the addition of the chase solution, samples were removed at 0, 10, 30 and 60 minutes. In all samples from OSH mutants, Hsp150p was secreted into the medium with equivalent kinetics to its wild-type control. Moreover, 60 minutes after pulse labeling and addition of chase solution no intracellular Hsp150p was detectable. In the exocytosis-defective sec1-1 strain scarcely any Hsp150p was secreted into the medium during the 60 minutes following pulse labeling and addition of chase solution, and Hsp150p was detected within cells (internalized Hsp150p appeared to degrade 60 minutes after the addition of chase solution but was nonetheless detectable). The autoradiograph of intracellular glycoproteins represented a longer exposure than that showing secreted extracellular glycoproteins.

View larger version (26K): [in a new window]   Fig. 5. Secretion of Gas1p to the plasma membrane was unaffected by OSH mutants. Autoradiograph of Gas1p immunoprecipitations from wild-type (W.T.; SEY6210), osh osh4-1 (CBY886), osh OSH4 (JRY6320) and sec18(ts) (JRY4130) cell lysates following SDS-PAGE. (p)Gas1p, precursor form of Gas1p; (m)Gas1p, mature plasma membrane form of Gas1p. After an incubation of 1 hour at 37°C, cells were pulse-labeled for 5 minutes and chased for the periods indicated (in minutes) at 37°C.

View larger version (65K): [in a new window]   Fig. 6. Temperature-sensitive OSH mutants were defective for lucifer yellow fluid-phase endocytosis. Lucifer yellow uptake and delivery to the vacuole was observed at 23°C, or after a 60 minute incubation at 37°C, in wild-type (Osh+; SEY6210), osh OSH4 (JRY6320), osh osh4-1 (CBY886) and osh osh4-2 (CBY892) cells. For comparison, lucifer yellow uptake was examined at 23°C in wild-type LAS17 (DDY904) and las17 (DDY1438) cells. In the wild-type Osh+ strain, vacuoles that were brightly stained with lucifer yellow were observed in 92% of cells grown at 23°C (65 out of 71 cells counted) and an equivalent percentage was seen at 37°C (91%; 64/70). In osh OSH4 cells, 68% exhibited brightly staining vacuoles at 23°C (69/101) and an equivalent percentage was observed at 37°C (69%; 69/99). In osh osh4-1 cells at 23°C, however, 42% exhibited bright lucifer yellow staining (36/85), and at 37°C, only 13% of cells (10/75) efficiently internalized lucifer yellow. Likewise, at 23°C 44% of osh osh4-2 cells (34/78) had bright fluorescent vacuoles and vacuolar remnants, and at 37°C, only 17% of these cells (13/79) were brightly stained. This result was comparable to that observed in the endocytosis mutant las17. Only 13% of las17 cells (15/116) were as brightly stained as in the wild-type parent, where 93% of the wild-type LAS17 cells (78/84) had efficiently absorbed lucifer yellow from the medium. All photographs represent equal exposures.

View larger version (59K): [in a new window]   Fig. 7. FM4-64 uptake, and Ste6p internalization and degradation, were inhibited in an OSH temperature-sensitive mutant. (A) Protein immunoblots of hemagglutinin (HA) epitope-tagged Ste6p expressed in osh osh4-1 cells (CBY966) and its congenic wild-type Osh+ parent (CBY968), and las17 cells (CBY1024) and its congenic wild-type LAS17 parent (CBY1026). In each of these strains, the stability of Ste6p was analyzed by adding cycloheximide to stop protein synthesis and then removing, at the times indicated, equal volumes of cell culture incubated at 37°C. Extracted proteins were separated on SDS gels and analyzed by immunoblot with antibodies that recognized the HA epitope. Note that in the wild-type strains, Ste6p-HA degradation occurred after 60 minutes but in the OSH temperature-sensitive mutant and las17 strain Ste6p-HA persisted beyond 90 minutes. All strains were incubated for 1 hour at 37°C before the addition of cycloheximide. (B) Wild-type cells (Osh+; SEY6210), osh osh4-1 (CBY926) and osh OSH4 (CBY924) cells were incubated for 30 minutes at 37°C, then treated with a short pulse of FM4-64 and chased with fresh medium. Cells were viewed by fluorescence microscopy 30 minutes after the FM4-64 pulse/chase (P/C) and photographs were taken with equal exposures.

View larger version (127K): [in a new window]   Fig. 10. ARV1 deletion mutants exhibited endocytosis defects and fragmented vacuoles. (A) Lucifer yellow (LY) uptake and delivery to the vacuole was observed in wild-type (W.T.; CBY858), erg2 (CBY678) and arv1 (CBY994) cells grown at 30°C. In the wild-type strain, brightly stained vacuoles were observed in 92% of cells (154 out of 167 cells counted). In erg2 cells, only 2% of cells efficiently internalized lucifer yellow (1/50), and 0% of arv1 cells exhibited brightly staining vacuole or vacuolar fragments (0/72). (B) Wild-type (CBY858), las17 (CBY1024) and arv1 (CBY994) were treated with a short pulse of FM4-64 and then chased with fresh medium (FM4-64 P/C). Cells cultured at 30°C were viewed by fluorescence microscopy 30 minutes after the FM4-64 pulse/chase (P/C) and photographs were taken with equal exposures. (C) Wild-type (CBY858) and arv1 (CBY994) cells were cultured at 30°C in the presence of FM4-64 for 4 hours to label all vacuoles and vacuolar fragments uniformly (FM4-64). In arv1 cells, vacuolar fragmentation was evident in the proliferation of FM4-64 staining vacuole-derived rings.

View larger version (81K): [in a new window]   Fig. 8. Filipin specifically stained yeast sterols. (A) Wild-type ERG9 (CBY858) and erg9 PMET3-ERG9 (CBY745) cells were treated with filipin. The left panels show cell morphology by DIC, and the right panels show the corresponding filipin staining observed by fluorescence microscopy. After addition of methionine, filipin fluorescence in erg9 PMET3-ERG9 cells was considerably less intense than in wild-type ERG9 cells. Photographs were taken with equivalent exposures, although with longer exposures background filipin fluorescence was detectable in the ERG9-repressed cells. (B) Sterol distribution in both arv1 (CBY994) and wild-type ARV1 (CBY858) cells were also examined using filipin fluorescence. In 41% of arv1 cells (52 out of 136 cells), fluorescence was observed within the cells whereas in wild-type cells, only 10% of cells (10/96) exhibited a comparable staining pattern. Arrows indicate filipin fluorescence associated with internal membranes.

View larger version (67K): [in a new window]   Fig. 9. Sterol lipid distribution was disrupted in OSH mutants. (A) The pattern of filipin fluorescence observed in wild-type cells (Osh+; SEY6210) was disrupted in osh OSH1 OSH3 (JRY6306) and osh OSH3 OSH6 (JRY6312) mutant cells. Bright fluorescence at the plasma membrane was observed in 98% of wild-type cells (54 out of 55 cells counted), 98% of osh OSH1 OSH3 cells (54/55), and in 66% of osh OSH3 OSH6 cells (19/34). Bright internal fluorescence was observed in 25% of wild-type cells (11/44), 36% of osh OSH1 OSH3 cells (19/53), and in 76% of osh OSH3 OSH6 cells (26/34). (B) osh PMET3-OSH2 (JRY6326) cells were cultured in the presence and absence of methionine and treated with filipin. In each series, left panels show cell morphology by DIC, and the right panels show the corresponding filipin fluorescence. Cells grown in the absence of methionine (-Met) displayed normal cell morphology and the pattern of filipin staining was comparable to that seen in wild-type cells. In the presence of methionine (+Met), Osh depleted osh PMET3-OSH2 cells were larger in size, bud morphology and septation were defective, and the intensity of intracellular filipin fluorescence increased. (C) Filipin fluorescence was examined in wild-type cells (SEY6210), and in the temperature-sensitive mutants osh osh4-1 (CBY926), and osh osh4-2 (CBY928). At 23°C, more intracellular filipin fluorescence was observed in the OSH mutants compared with wild-type cells, and after 60 minutes at 37°C the intensity of intracellular fluorescence increased in mutant cells. In both (B) and (C), images of filipin fluorescence do not represent equal exposures; exposures were longer for wild-type cells.

View larger version (99K): [in a new window]   Fig. 11. Sterol lipid distribution was unaffected in END4/SLA2 temperature-sensitive mutants. Leftmost panels show whole cell morphology by DIC, and the rightmost panels show the same cells stained with filipin. No intracellular accumulation of sterols was detectable by filipin staining in sla2/end4-1 cells (RH286-1C) as compared with wild type (W.T.; W303-1A).

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