A block in endoplasmic reticulum-to-Golgi trafficking inhibits phospholipid synthesis and induces neutral lipid accumulation

Abstract

Seeking to better understand how membrane trafficking is coordinated with phospholipid synthesis in yeast, we investigated lipid synthesis in several Sec(-) temperature-sensitive mutants, including sec13-1. Upon shift of sec13-1 cells to the restrictive temperature of 37 degrees C, phospholipid synthesis decreased dramatically relative to the wild type control, whereas synthesis of neutral lipids, especially triacylglycerol (TAG), increased. When examined by fluorescence microscopy, the number of lipid droplets appeared to increase and formed aggregates in sec13-1 cells shifted to 37 degrees C. Electron microscopy confirmed the increase in lipid droplet number and revealed that many were associated with the vacuole. Analysis of lipid metabolism in strains lacking TAG synthase genes demonstrated that the activities of the products of these genes contribute to accumulation of TAG in sec13-1 cells after the shift to 37 degrees C. Furthermore, the permissive temperature for growth of the sec13-1 strain lacking TAG synthase genes was 3 degrees C lower than sec13-1 on several different growth media, indicating that the synthesis of TAG has physiological significance under conditions of secretory stress. Together these results suggest that following a block in membrane trafficking, yeast cells channel lipid metabolism from phospholipid synthesis into synthesis of TAG and other neutral lipids to form lipid droplets. We conclude that this metabolic switch provides a degree of protection to cells during secretory stress.

FIGURE 1.

Metabolic pathways for the synthesis of phospholipids and neutral lipid classes in S. cerevisiae. PA serves as a precursor to CDP-DAG, precursor to PI and PS, which serves as precursor to PE. PC is synthesized either via methylation of PE or from CDP-choline and DAG via the Kennedy pathway. DAG, produced by dephosphorylation of PA, serves as a precursor to TAG, as well as PC via the Kennedy pathway. When PC is synthesized via the Kennedy pathway, the ³²P label enters via phosphorylation of choline. In contrast, when PC is synthesized via methylation of PE, ³²P is derived from PA. Metabolites in boxes denote key intermediaries and/or end products that are relevant in the discussion of this study. Metabolites in ovals are derived from PA. The bold arrows indicate the multiple steps of fatty acid synthesis, and the block arrows indicate the mevalonate pathway for the synthesis of ergosterol. The asterisks indicate the two places where ³²P label enters into the pathways for the synthesis of the major phospholipids shown here.

FIGURE 2.

Pulse labeling of phospholipids with ³²P in wild type (wt) and sec13-1 cells following a shift from 25 °C to the sec13-1 restrictive temperature of 37 °C. Cells were grown in I⁺ medium at 25 °C until mid-logarithmic phase of growth (A_{600 nm} = 0.5). One aliquot was maintained at 25 °C, and the reminder was shifted to 37 °C. A 5-ml sample of each culture was maintained at 25 °C and immediately labeled with 100 μCi/ml [³²P]orthophosphate for 20 min (0 min time point). Samples from the cultures shifted to 37 °C were labeled for 20 min at 60, 120, and 180 min after the temperature shift. Lipids were extracted and analyzed as described under “Experimental Procedures.” Data are expressed as counts of ³²P radiolabel incorporated into total and individual phospholipids per A_{600 nm} unit in the cell culture. A, ³²P label associated with phosphatidic acid (PA). B, ³²P label associated with TPL = total label associated with the sum of the following phospholipids: PI, PS, PE, and PC. The dotted line traces the increased rate of PI synthesis in wild type cells and the decrease in PI synthesis in sec13-1 cells, following the shift to 37 °C.

FIGURE 3.

Comparison of CPY processing in sec13-1 mutant strain at permissive and restrictive temperatures. sec13-1 cells were preincubated for 15 min at 25 °C (permissive temperature) or 37 °C (restrictive temperature), pulse-labeled with [³⁵S]methionine/cysteine for 5 min, and chased with an excess of cold methionine and cysteine at 25 or 37 °C. Cell lysates from samples collected at indicated time points were immunoprecipitated with anti-CPY antibodies followed by SDS-PAGE analysis and autoradiography. Migration of ER-glycosylated (p1) and Golgi-modified (p2) precursors and vacuolar mature form (mCPY) are indicated.

FIGURE 4.

Splicing of HAC1 mRNA is initiated in the sec13-1 mutant shortly after elevation to 37 °C. Strains were grown to mid-logarithmic growth phase in I⁺ medium at 25 °C. RNA was isolated from yeast cells immediately prior to and following temperature shift to 37 °C at indicated time points. HAC1 transcript abundance in both wild type and sec13-1 cells was analyzed by Northern blotting. The appearance of the spliced HAC1ⁱ form indicates activation of the UPR pathway. Ethidium bromide staining of 25 S and 18 S rRNA served as loading controls (not shown).

FIGURE 5.

Alteration in total lipid composition of wild type, sec13-1, and sec31-2 and sec6-4 cells following a shift to 37 °C. Cells were labeled to steady state with [1-¹⁴C]acetate (1 μCi/ml) in I⁺ medium at 25 °C. Cultures were allowed to grow to mid-logarithmic phase and were then shifted to 37 °C for 180 min. Samples were taken at 0, 60, 120, and 180 min after the elevation of temperature. The results represent the average of two independent experiments for sec31-2 and sec6-4. The data for wild type and sec13-1 represent the average of four and three independent experiments, respectively. Experimental error was less than 10% in all cases. Error bars are not shown for clarity of presentation. The lipids indicated are as follows: PI, solid squares; PC, solid circles; PS, open circles; PE, open triangles; NL, open diamonds. A, wild type cells; B, sec13-1; C, sec31-2 cells; D, sec6-4 cells.

FIGURE 6.

Effect of high temperature on the neutral lipid content of wild type, sec13-1, sec31-2, and sec6-4 cells. The cells were grown and labeled to steady state with [1-¹⁴C]acetate (1 μCi/ml), as described in Fig. 5. Experimental error was less than 10%. (Error bars are not shown.) The lipids indicated are as follows: free fatty acids, solid squares; triacylglycerols, open triangles; free sterols, open circles; diacylglycerols, solid circles; sterol esters, open diamonds. A, wild type cells; B, sec13-1; C, sec31-2 cells; D, sec6-4 cells. (The data for wild type and sec13-1 are also repeated in Fig. 12 on expanded scales.)

FIGURE 7.

Fluorescence microscopic analysis of lipid droplets. Prior to microscopy, cultures were grown in I⁺ medium at 25 °C as described under “Experimental Procedures” and were then diluted to A_{600 nm} = 0.1 in 2 ml of I⁺ medium in multiwell plates, and incubated at 25 °C in an Eppendorf incubator/mixer. After reaching A_{600 nm} = 0.5, cultures were shifted to 37 °C, and aliquots removed at time 0 and 120 min, fixed and stained with BODIPY® 493/503, and analyzed by fluorescence microscopy. a, wild type cells at 25 °C (zero time at 37 °C); b, after 120 min of incubation at 37 °C; c, sec13-1 cells at 25 °C (zero time at 37 °C); d, after 120 min of incubation at 37 °C; e, sec13-1lro1Δdga1Δare2Δ cells at 25 °C (zero time at 37 °C); and f, after 120 min incubation at 37 °C. Note that fluorescence emitted from BODIPY® that is dissolved in membrane aggregates, as opposed to lipid droplets, is red shifted, as indicated by a more yellowish fluorescence in e and f. Insets are 2-fold magnifications of representative cells. Bar = 5 μm.

FIGURE 8.

Flow cytometric analysis of lipid droplet content of wild type (wt), sec13-1, sec31-2, and sec6-4 cells at 25 and 37 °C. Yeast cells were grown until mid-logarithmic phase in I⁺ medium at 25 °C and split in two cultures; one of the cultures was shifted to 37 °C, and the other one was kept at 25 °C as a control. After 120 min of incubation, samples were withdrawn and fixed with 3.7% formaldehyde. Formaldehyde-fixed cells were permeabilized by treatment with 0.1% Triton X-100 in 1× PBS buffer, pH 8, for 10 min in and incubated with 10 μm BODIPY® 493/503 for 15 min to label the lipid droplets and were analyzed as described under “Experimental Procedures” by flow cytometer. Open bars illustrate the strains incubated at 25 °C; solid bars represent the strains incubated at 37 °C. The results represent the mean of three independent experiments.

FIGURE 9.

Electron microscopy reveals lipid droplets associated with vacuoles in sec13-1 cells. Cells were grown as in Fig. 6, and then processed for transmission electron microscopy as described under “Experimental Procedures.” A, wild type (WT) cells at 25 °C; B, wild type cells after 120 min at 37 °C; C, sec13-1 cells at 25 °C; D-F, sec13-1 cells after 120 min at 37 °C. Arrows point to lipid droplets adjacent to the ER (A-C and E) or tightly associated with vacuoles (D and F). The arrowhead in F points to electron lucent glycogen clusters found in sec13-1 cells after 120 min at 37 °C. Vac = vacuole. Bars = 0.5 μm.

FIGURE 10.

Higher magnification views of sec13-1 cells after 120 min at 37 °C reveal lipid droplets in close association with the vacuole. Electron microscopy performed as in Fig. 9. Lipid droplets are seen in association with the vacuoles (arrows in A-D), and in some cases were apparently being internalized into the vacuole (arrows in C and D). Also, the electron lucent clusters of glycogen (arrowheads) are interconnected and found under the cortical ER (E and F). Vac = vacuole; bars = 0.2 μm.

FIGURE 11.

Lugol staining of wild type and sec13-1 strains grown at 37 °C for 2 h. Cells were grown at 25 °C in I⁺ medium and then shifted to 37 °C prior to microscopy as described in Fig. 7 and stained as described under “Experimental Procedures.” a, wild type cells; b, sec13-1 cells. Bars = 5 μm.

FIGURE 12.

Deletion of the LRO1, DGA1, and ARE2 genes in the sec13-1 genetic background results in a decrease in accumulation of neutral lipids, compared with the sec13-1 parent, following elevation to 37 °C. Cells were grown as described for steady state labeling in the presence of 1 μCi/ml [1-¹⁴C]acetate in I⁺ medium at 25 °C as described under “Experimental Procedures” and Figs. 5 and 6. At the mid-logarithmic phase of growth, the cultures were shifted to 37 °C. Samples were taken for lipid analysis at 0, 60, 120, and 180 min after the shift to 37 °C. Note that the data for wild type and sec13-1 are identical to (but are displayed on a different scale) the data shown in Fig. 6. Data are representative of at least two independent experiments. Experimental error was less than 10%. Error bars are not shown for clarity of presentation. The neutral lipids depicted are as follows: free fatty acids, solid squares; triacylglycerols, open triangles; free sterols, open circles; diacylglycerols, solid circles. Note that the free sterols (open circles) are depicted on a different scale than the other lipids.

FIGURE 13.

Deletion of the LRO1, DGA1, and ARE2 genes in the sec13-1 genetic background leads to an increase in PC and PI content, relative to the sec13-1 parent strain, following a shift to 37 °C. Changes in the accumulation of phospholipids in response to an increase in temperature in wild type and sec13-1 cells carrying deletions in some steps of the TAG formation was assessed by labeling the cells in the presence of [1-¹⁴C]acetate under conditions identical to those described in Figs. 6, 7, and 12. Experimental error was less than 10%. Error bars are not shown for clarity of presentation. The lipids indicated are as follows: PI, solid squares; PC, solid circles; PS, open circles; PE, open triangles.

FIGURE 14.

The lro1Δ and dga1Δ mutations lower the permissive temperature of strains carrying the sec13-1. Overnight cultures were diluted to A_{600 nm} = 0.1 in 25 ml of YPD medium and allowed to grow to mid-logarithmic phase at 25 °C. Each sample was diluted in multiwell plates by 1:10 serial dilutions, and 4 μl of cells from each dilution were spotted on YPD plates and allowed to grow at the designated temperatures for 2 days.

FIGURE 15.

The lro1Δ, dga1Δ, and are2Δ mutations lower the permissive temperature of strains carrying the sec13-1 mutation on synthetic complete medium lacking inositol. Overnight, cultures were diluted to A_{600 nm} = 0.1 in 25 ml of I⁺ or I^- medium and allowed to grow to mid-logarithmic phase at 25 °C. Each sample was diluted in multiwell plates by 1:10 serial dilutions, and 10 μl of cells from each dilution were spotted on I⁺ and I^- plates and allowed to grow at the designated temperatures for 2 days.