Tsc13p colocalizes and copurifies with Nvj1p in the perinuclear ER independent of Vac8p. (A) Plasmid-bound Tsc13p-EYFP expressed from the inducible CUP1 promoter (P_CUP1-TSC13-EYFP) accumulates at NV junctions in wt cells. Vacuolar membranes (red) and nuclear chromatin (blue) were costained with FM4-64 and Hoechst, respectively. NV junctions (see arrows) look yellow in the overlay. (B) The accumulation of Tsc13p-EYFP at NV junctions is dependent on both Nvj1p and Vac8p. nvj1-Δ cells and vac8-Δ cells expressing P_CUP1-TSC13-EYFP were analyzed at log phase by confocal microscopy. Vacuolar membranes (red) and nuclear chromatin (blue) were costained as described above. Arrows point out the novel accumulation of Tsc13p-EYFP at patches in the perinuclear ER of vac8-Δ cells. (C) Overexpression of Nvj1p-Fc in nvj1-Δ cells restores the targeting of Tsc13p-EYFP to NV junctions and targets Tsc13p-EYFP to perinuclear ER patches in vac8-Δ cells. Log phase nvj1-Δ and vac8-Δ cells harboring both P_CUP1-TSC13-EYFP and P_CUP1-NVJ1-Fc were induced for 1.5 h with CuSO₄. Vacuolar membranes (red) and nuclear chromatin (blue) were stained as described above. NV junctions look yellow in the overlay (i, see arrow). Tsc13p-EYFP accumulates at nonyellow nuclear ER patches in vac8-Δ cells after Nvj1p-Fc induction (ii, see arrow). (D) Tsc13p-EYFP specifically copurifies with Nvj1p-Fc from nvj1-Δ and vac8-Δ cell extracts. Lysates of cells harboring P_CUP1-TSC13-EYFP and either P_CUP1-NVJ1-Fc or empty vector were prepared as described in Materials and Methods. Nvj1p-Fc complexes were purified using protein A-conjugated agarose beads. The copurification of Tsc13p-EYFP was assessed by immunoblot using polyclonal GFP antibodies.

Cerulenin perturbs the targeting of Tsc13p to NV junctions and stunts PMN morphology in starved cells. (A and B) Localization of chromosomally expressed cTsc13p-EGFP (A) or cNvj1p-EYFP (B) to NV junctions and PMN blebs or vesicles in cells starved overnight in the presence or absence of cerulenin, myristic acid, or myriocin (see Materials and Methods). Nuclear chromatin (blue) was stained with Hoechst to reveal the position of nuclei in the overlays. Arrowheads point to stunted PMN blebs and vesicles in cerulenin-treated cells as revealed by both cTsc13p-EGFP (A) and cNvj1p-EYFP (B) fluorescence. Furthermore, cTsc13p-EGFP, which normally accumulates at NV junctions in starving cells treated with ethanol (A), looks delocalized from NV junctions in cerulenin-treated cells but reaccumulates at NV junctions upon supplementation with myristic acid (C₁₄). In contrast, cNvj1p-EYFP localizes strictly to NV junctions after all treatments (B). PMN structures in myriocin-treated cells or cerulenin-treated cells incubated with myristic acid (C₁₄) are of similar size and shape as mock-treated cells.

PMN morphology is significantly altered in VLCFA elongation mutants. (A) The size and lumenal diameter of PMN blebs and vesicles are significantly undersized in tsc13-1 and elo3-Δ tsc13-1 fatty acid elongation mutants during starvation. PMN morphology was analyzed in tsc13-1, elo3-Δ tsc13-1, and their wt parental strain (TDY2037) using P_CUP1-NVJ1-EYFP as a reporter. Cells were grown to ∼0.1 OD₆₀₀ and starved in SD-N for 3.5 h before confocal imaging. Vacuolar membranes (red) and nuclear chromatin (blue) were stained with FM4-64 and Hoechst, respectively, and superimposed. PMN diameters were measured using the Profile function of the confocal software (see Materials and Methods). (B) The mean diameters of tsc13-1 and elo3-Δ tsc13-1 PMN structures are statistically and incrementally smaller than wt cells but become enlarged upon the introduction of plasmid-encoded TSC13. Lumenal diameters (micrometers) of the largest representative PMN structures were measured in 10–15 cells from starving wt, tsc13-1, and elo3Δ tsc13-1 cultures, respectively, using the Profile function of the confocal software (see Materials and Methods). Cells were cultured, labeled, and starved as described in A. These experiments were then repeated in tsc13-1 and elo3Δ tsc13-1 cells harboring pRS425-TSC13, and the values were averaged.

Tsc13p is sequestered from peripheral and nuclear ER pools to NV junctions in an Nvj1p-dependent manner. (A) Localization of chromosomally expressed cTsc13p-EGFP as a function of NVJ1 induction. Cells expressing cTsc13p-EGFP and harboring either P_CUP1-NVJ1 or empty vector were analyzed at low OD₆₀₀ (∼0.3). P_CUP1-NVJ1 expression was maintained at a basal level (iii and iv) or induced for 1.5 h with CuSO₄ (v and vi) similar to empty vector control cells (i and ii). Nuclear chromatin (blue) and vacuolar membranes (red) were costained with Hoechst and FM4-64, respectively. NV junctions look yellow in the overlay (bottom). The fluorescence of cTsc13p-EGFP decreases at peripheral ER pools (i) and perinuclear ER pools (iii) but increases at NV junctions (v and vi) as Nvj1p levels rise. (B) Immunoblot analysis of cTsc13p-EGFP protein levels in whole cell extract as a function of NVJ1 expression. Wild-type extract lacking cTsc13p-EGFP denoted as N. (C) Associate ER proteins involved in VCLFA biosynthesis (Elo2p, Elo3p, and YBR159p) do not specifically accumulate at NV junctions when NVJ1 is induced (nor are they excluded). Cells harboring P_CUP1-NVJ1 and expressing chromosomally expressed cElo3p-EGFP, cYbr159p-EGFP, or plasmid-encoded Elo2p-EGFP were analyzed at log phase and stained for nuclear chromatin (blue) or vacuolar membranes (red) as described above.

Tsc13p accumulates in nuclear PMN blebs and vesicles during stationary phase and nitrogen starvation. (A and B) Tsc13p-EYFP localizes to PMN blebs and vesicles in wt (A) and atg7-Δ (B) cells upon nitrogen starvation. Cells harboring P_CUP1-TSC13-EYFP were grown in SCGlu to stationary phase (∼1.0 OD₆₀₀), shifted to SD-N medium, and analyzed by confocal microscopy after 3 h of starvation. Vacuolar membranes (red) and nuclear chromatin (blue) were stained with FM4-64 and Hoechst, respectively, and superimposed. Tsc13p-EYFP localizes to yellow-labeled PMN structures (see arrows) independent of ATG7. (C) Chromosomally expressed cTsc13p-EGFP increasingly accumulates in PMN structures proportional to NVJ1 expression. Cells expressing cTsc13p-EGFP and harboring P_CUP1-NVJ1 (iii and iv) or empty vector (i and ii) were grown to low OD₆₀₀ (∼0.3), shifted to SD-N media, and analyzed by confocal microscopy after 3 h of starvation. Vacuolar membranes (red) and nuclear chromatin (blue) were stained as described above. PMN structures (see arrows) look yellow in the overlay and increase in size proportional to the targeting of cTsc13p-EGFP to NV junctions and NVJ1 expression.

Vac8p membrane association as well as PMN-mediated protein turnover are normal in the presence of cerulenin. (A) Vac8p efficiently fractionates with isolated membranes in cells starved in the presence or absence of cerulenin. Whole cell extracts were fractionated by differential centrifugation as described in Materials and Methods. As a control, vac8-4 cells (expressing a mutant form of Vac8p lacking myristoylation and palmitoylation sites) were fractionated in a similar manner. Vac8p was detected in isolated membrane fractions (P13 and P100) but not soluble fractions (S100) of extracts from mock-treated and cerulenin-treated cells. (B) The starvation-induced degradation of Nvj1p-myc and cTsc13p-EGFP is normal in the presence of cerulenin. A “pulse” of Nvj1p-myc was expressed in wt cells harboring P_GAL1-NVJ1-myc, and its degradation was analyzed after glucose-induced repression in nitrogen starvation media, which was supplemented with cerulenin or ethanol (see Materials and Methods). The expression of cTsc13p-EGFP was controlled from the TSC13 chromosomal locus. Wild-type control extract denoted as C.