Integral membrane proteins Brr6 and Apq12 link assembly of the nuclear pore complex to lipid homeostasis in the endoplasmic reticulum

Abstract

Cells of Saccharomyces cerevisiae lacking Apq12, a nuclear envelope (NE)-endoplasmic reticulum (ER) integral membrane protein, are defective in assembly of nuclear pore complexes (NPCs), possibly because of defects in regulating membrane fluidity. We identified BRR6, which encodes an essential integral membrane protein of the NE-ER, as a dosage suppressor of apq12 Delta. Cells carrying the temperature-sensitive brr6-1 allele have been shown to have defects in nucleoporin localization, mRNA metabolism and nuclear transport. Electron microscopy revealed that brr6-1 cells have gross NE abnormalities and proliferation of the ER. brr6-1 cells were hypersensitive to compounds that affect membrane biophysical properties and to inhibitors of lipid biosynthetic pathways, and displayed strong genetic interactions with genes encoding non-essential lipid biosynthetic enzymes. Strikingly, brr6-1 cells accumulated, in or near the NE, elevated levels of the two classes of neutral lipids, steryl esters and triacylglycerols, and over-accumulated sterols when they were provided exogenously. Although neutral lipid synthesis is dispensable in wild-type cells, viability of brr6-1 cells was fully dependent on neutral lipid production. These data indicate that Brr6 has an essential function in regulating lipid homeostasis in the NE-ER, thereby impacting NPC formation and nucleocytoplasmic transport.

Fig. 1.

Dosage suppression of growth, mRNA export and nucleoporin mislocalization of brr6-1 and apq12Δ cells by APQ12 and BRR6. (A) Serial dilutions of apq12Δ and brr6-1 cells overexpressing BRR6 and APQ12 were spotted onto plates and incubated at the temperatures shown. (B) In situ hybridization assay to analyze the effect on the mRNA-export defects of apq12Δ and brr6-1 cells of over-expressing BRR6 and APQ12 by using multicopy plasmids. (C) Direct visualization of Nup82-GFP and Nup188-GFP in apq12Δ and brr6-1 cells overexpressing BRR6 and APQ12. Nup82-GFP and Nup188-GFP were expressed from the NUP82 and NUP188 chromosomal loci. Scale bars: 5 μm.

Fig. 2.

Mislocalization of cytoplasmic filament nucleoporins in brr6-1 cells. (A) Wild-type and brr6-1 cells expressing Nup60-GFP or Nup82-GFP were shifted to 20°C overnight and live cells directly visualized. (B) Wild-type and brr6-1 cells grown either at 30°C or shifted to 18°C overnight before visualization. Note the heterogeneity of the distribution of Nup82-GFP in brr6-1 but not in wild-type cells (right). Scale bars: 5 μm.

Fig. 3.

Electron microscopy reveals NE abnormalities in brr6-1 cells. Wild-type and brr6-1 cells were grown at 30°C or shifted to 18°C overnight and processed for thin-section electron microscopy. (A) Wild-type cells, 18°C; (B) wild-type cells, 30°C; (C-E) brr6-1 cells, 18°C; (F,G) brr6-1 cells, 30°C. Arrows and arrowheads: electron-dense structures and inclusions [arrowheads (A,B,G), arrow (D)], sometimes clustered [arrows (E,F)] or clustered in additional double membranes [black arrows (C,G)]; extensive white expansions of the NE and ER [white arrows (C)]. n, nucleus.

Fig. 4.

Effect of compounds that alter membrane properties or inhibit lipid biosynthetic pathways on growth of wild-type, brr6-1 and apq12Δ cells. Serial dilutions of wild-type, brr6-1 and apq12Δ cells were grown on YPD plates or on plates containing: (A) the membrane fluidizers benzyl alcohol or oleic acid, or (B) drugs that inhibit sterol synthesis (terbinafine and ketoconazole), or fatty acid synthesis (cerulenin) and incubated at the temperatures shown.

Fig. 5.

Analysis of TAGs and sterols in apq12Δ and brr6-1 cells. (A) brr6-1 cells accumulate triacylglycerols. Cells were labeled with [³H]palmitic acid for 6 hours at 37°C. Lipids were extracted, separated by thin layer chromatography and levels of radiolabeled fatty acids in the different lipid classes were quantified by radio-scanning. (B) brr6-1 cells have elevated levels of steryl esters. (C) brr6-1 and apq12 Δ cells accumulate the sterol precursor episterol. Cells were cultivated overnight at 24°C. Lipids were extracted and sterols were analyzed and quantified by GC-MS, using cholesterol as an internal standard. These analyses were performed independently two (B,C) or three (A) times and results shown are means ± s.e.m.

Fig. 6.

Overexpression of BRR6 suppresses the elevated level of episterol in apq12Δ cells. apq12Δ and brr6-1 cells containing an empty vector or overexpressing BRR6 (A) or APQ12 (B), respectively, from multicopy plasmids, were cultivated overnight at 24°C. Lipids were extracted and sterols were analyzed and quantified by GC-MS. These analyses were performed independently two times and results shown are means ± s.e.m.

Fig. 7.

Production of steryl esters and triacylglycerides is essential in brr6-1 cells. (A) Analysis of brr6-1 synthetic interactions with are1 Δ are2 Δ. ARE1 and ARE2 were deleted in a brr6-1 background to produce the are1 Δ are2 Δ brr6-1 triple mutant. Growth of the indicated strains on YPD plates is shown. (B) Analysis of brr6-1 synthetic interactions with lro1 Δ dga1 Δ. LRO1 and DGA1 were deleted in a brr6-1 strain containing a wild-type copy of BRR6 on a URA3 plasmid. Cells were plated on YPD plates and then replicated to YPD plates and to plates containing 5-fluoro-orotic acid (5-FOA) to select for colonies that have lost the URA3 BRR6 plasmid. Plates were incubated for 4 days at 24°C.

Fig. 8.

brr6-1 cells accumulate free sterols and steryl esters. (A) Subcellular distribution of NBD-cholesterol. Heme-deficient cells of the indicated genotype and expressing the ER marker Kar2-mRFP-HDEL were incubated with the fluorescent sterol analog NBD-cholesterol for 1 hour at 24°C and the distribution of NBD-cholesterol was analyzed by fluorescence microscopy. White arrowheads indicate staining of the NE-ER. The numbers indicate the proportion of NBD-cholesterol-stained lipid droplets that colocalize with the Kar2-mRFP-HDEL-stained NE-ER (n=100 cells). Scale bar: 5 μm. (B) Heme-deficient cells of the indicated genotype were incubated with [¹⁴C]cholesterol. Samples were removed at the time points indicated, lipids were extracted and the levels of free and esterified cholesterol were quantified. Results shown are means ± s.e.m.

Fig. 9.

brr6-1 cells have aberrant lipid droplets. Cells were transformed with a plasmid expressing the lipid droplet marker protein Erg6-RFP and analyzed by fluorescence microscopy. White arrowheads indicate lipid droplets that encircle the NE-ER. The numbers indicate the proportion of cells that display circular arrangement of Erg6-RFP marked lipid droplets around the nucleus (n=100 cells). DNA was revealed by staining with DAPI. Scale bar: 5 μm.