An essential nuclear envelope integral membrane protein, Brr6p, required for nuclear transport

Abstract

Despite rapid advances in our understanding of the function of the nuclear pore complex in nuclear transport, little is known about the role the nuclear envelope itself may play in this critical process. A small number of integral membrane proteins specific to the envelope have been identified in budding yeast, however, none has been reported to affect transport. We have identified an essential gene, BRR6, whose product, Brr6p, behaves like a nuclear envelope integral membrane protein. Notably, the brr6-1 mutant specifically affects transport of mRNA and a protein reporter containing a nuclear export signal. In addition, Brr6p depletion alters nucleoporin distribution and nuclear envelope morphology, suggesting that the protein is required for the spatial organization of nuclear pores. BRR6 interacts genetically with a subset of nucleoporins, and Brr6-green fluorescent protein (GFP) localizes in a punctate nuclear rim pattern, suggesting location at or near the nuclear pore. However, Brr6-GFP fails to redistribute in a (Delta)nup133 mutant, distinguishing Brr6p from known proteins of the pore membrane domain. We hypothesize that Brr6p is located adjacent to the nuclear pore and interacts functionally with the pore and transport machinery.

Fig. 1. The brr6-1 mutant accumulates bulk poly(A) RNA in the nucleus and at the nuclear rim. Shown are the mRNA localization patterns in BRR6 and brr6-1 cells at 30°C determined by in situ hybridization with a digoxygenin-labeled oligo dT50 probe. DAPI staining was used to confirm the locations of the cell nuclei. The inserts in the brr6-1 panels show dT50 and DAPI staining of a single nucleus enlarged to show the mRNA accumulation at the nuclear rim evident in some mutant cells. Bar, 10 µm.

Fig. 2. The brr6-1 mutation causes a defect in NES protein export. Shown are the localization patterns of NLS/NES(GFP)₂ and NLS(GFP)₃ reporters in BRR6 and brr6-1 cells as indicated. The NLS/NES construct accumulates at the nuclear rim in brr6-1 as confirmed by DAPI staining (data not shown). The NLS construct shows nucleoplasmic staining in both mutant and wild-type cells. Bar, 10 µm.

Fig. 3. Brr6-GFP localizes to the nuclear rim. (A) The localization patterns of a Brr6-GFP fusion protein expressed from a low copy (CEN/ARS) vector or a high copy 2 µm plasmid as indicated. At low copy, Brr6-GFP shows predominantly punctate nuclear rim staining. Upon overexpression, Brr6-GFP accumulates at the nuclear and cell peripheries. (B) The effect of BRR6 (BRR6/pRS424) overexpression relative to the empty vector (pRS424) on growth of wild-type (W303), Δnup116::HIS3, Δnup1::HIS3 and Δnup188::HIS3 strains (all W303 strain background) on -TRP medium. BRR6 overexpression causes dramatic growth defects in Δnup1 and Δnup188 cells with only mild effects on wild-type and Δnup116 cells. (C) Nup188-GFP and Brr6-GFP (Brr6-GFP/pRS424) localization patterns in wild-type (RS456) and Δnup133 cells. In contrast to Nup188-GFP, Brr6-GFP fails to show clustering in the Δnup133 mutant. Bar, 10 µm.

Fig. 4. The extraction profile of Brr6p from crude lysates typifies an integral membrane protein. Shown are analyses of Brr6-GFP (A), Pom152 (B) and Nup188-HA (C) extracted from whole-cell lysates using four different extraction conditions: (i) 150 mM NaCl; (ii) 1 M NaCl; (iii) 150 mM NaCl, 100 mM sodium carbonate; (iv) 150 mM NaCl, 4% Triton X-100. Supernatant (S) and pellet (P) lanes are indicated. Western blots were probed with antibodies against GFP, Pom152 or the HA tag. Brr6-GFP and Pom152 are present in the supernatant only after detergent treatment, while Nup188-HA is also evident in high salt and high pH supernatants.

Fig. 5. Trypsin digestion yields a detergent-sensitive C-terminal Brr6 fragment. (A) The BRR6 ORF, showing the locations of the brr6-1 mutation (asterisk), the KAP123 homology and the two predicted transmembrane domains. A sequence alignment of the homologous portions of BRR6 and KAP123 is shown. No significant similarity was found between BRR6 and the karyopherin β family prototype, KAP95. (B) The results of trypsin digestion experiments carried out on whole-cell lysates containing Brr6-GFP and polyoma-Brr6 constructs tagged at the C- and N-termini, respectively. Western blot analyses using antibodies against the GFP or polyoma tags demonstrate the protection of a C-terminal Brr6-GFP fragment (asterisk), which is sensitive to detergent. No protected N-terminal fragments are observed. (C) The likely topology of Brr6p in the membrane. The ends of the region of KAP123 homology (arrows) and the site of the brr6-1 mutation (asterisk) are indicated.

Fig. 6. Depletion of Brr6p causes redistribution of nucleoporins. (A) Nup188-GFP and Nsp1p localizations under conditions where Brr6p expressed from the GAL1-GAL10 promoter (galBRR6-Myc) was depleted by glucose inhibition. Nup188-GFP was assayed in living cells and Nsp1p was detected by indirect immunofluorescence in fixed cells. Growth in glucose medium for 5 h resulted in a consolidation of the Nup188-GFP and Nsp1p signals (examples indicated with arrows) in the galBRR6-Myc strain but not in BRR6-Myc in which BRR6 is under the control of its own promoter. (B) Nup188-GFP and Nsp1p distributions in BRR6 and brr6-1 cells. Bar, 10 µm.

Fig. 7. Brr6p depletion and the brr6-1 mutation cause changes in NE morphology. Shown are transmission electron microscope images of thin sections through cells prepared by high-pressure freezing. (A) A single example of BRR6-Myc and two examples of galBRR6-Myc cells grown in galactose medium and then switched to glucose medium for 5 h. The upper panels show examples of whole nuclei (NU) (bar, 500 nm). Examples of typical pores are marked with an asterisk (*) and are shown at higher magnification in the lower panels (bar, 100 nm). (B) Images of BRR6 and brr6-1 cells grown in glucose at 30°C. Upper panels show whole nuclei (bar, 500 nm), lower panels show individual pores (*) at higher magnification (bar, 100 nm).