text: The integral membrane protein snl1p is genetically linked to yeast nuclear pore complex function. - PubMed - NCBI
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  See comment in PubMed Commons belowMol Biol Cell. 1998 Feb;9(2):355-73.The integral membrane protein snl1p is genetically linked to yeast nuclear pore complex function.Ho AK1, Raczniak GA, Ives EB, Wente SR.Author information1Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.AbstractIntegral membrane proteins are predicted to play key roles in the biogenesis and function of nuclear pore complexes (NPCs). Revealing how the transport apparatus is assembled will be critical for understanding the mechanism of nucleocytoplasmic transport. We observed that expression of the carboxyl-terminal 200 amino acids of the nucleoporin Nup116p had no effect on wild-type yeast cells, but it rendered the nup116 null strain inviable at all temperatures and coincidentally resulted in the formation of nuclear membrane herniations at 23 degrees C. To identify factors related to NPC function, a genetic screen for high-copy suppressors of this lethal nup116-C phenotype was conducted. One gene (designated SNL1 for suppressor of nup116-C lethal) was identified whose expression was necessary and sufficient for rescuing growth. Snl1p has a predicted molecular mass of 18.3 kDa, a putative transmembrane domain, and limited sequence similarity to Pom152p, the only previously identified yeast NPC-associated integral membrane protein. By both indirect immunofluorescence microscopy and subcellular fractionation studies, Snl1p was localized to both the nuclear envelope and the endoplasmic reticulum. Membrane extraction and topology assays suggested that Snl1p was an integral membrane protein, with its carboxyl-terminal region exposed to the cytosol. With regard to genetic specificity, the nup116-C lethality was also suppressed by high-copy GLE2 and NIC96. Moreover, high-copy SNL1 suppressed the temperature sensitivity of gle2-1 and nic96-G3 mutant cells. The nic96-G3 allele was identified in a synthetic lethal genetic screen with a null allele of the closely related nucleoporin nup100. Gle2p physically associated with Nup116p in vitro, and the interaction required the N-terminal region of Nup116p. Therefore, genetic links between the role of Snl1p and at least three NPC-associated proteins were established. We suggest that Snl1p plays a stabilizing role in NPC structure and function.PMID: 9450961 PMCID: PMC25263 [Indexed for MEDLINE] Free PMC ArticleShareImages from this publication.See all images (11)Free textFigure 1The nup116-C lethal phenotype. (A) Schematic diagrams of the structural regions of Nup116p, Nup100p, and Nup145p and their related deletion/truncation polypeptides as referred to in this study. The N-terminal region of Nup116p spans amino acid residues 1–180, GLFG region residues 181–725, the C-terminal region residues 726-1113, the CN region residues 726–919, and the CC region residues 914-1113. The similarity between the C-terminal regions of Nup116p and Nup100p is 78%, whereas among Nup116-C, Nup100-C, and the middle region of Nup145 (Nup145-M) it is 55% (; ; ). The NRM region in Nup116p spans residues 998-1005 (). FL, full- length protein. (B) The nup116Δ strain is inviable when the homologous Nup116-C, Nup100-C, and Nup145-M regions fused to GST are expressed from a galactose-inducible promoter. The plasmids expressing the indicated protein regions (see Table ) were transformed into W303α (NUP116) or SWY27 (nup116Δ::HIS3) (shaded sectors). Strains were grown at 23°C for 7 d on SM-trp, 2% glucose, or 2% galactose. Expression of Nup116-C, Nup100-C, and Nup145-M inhibited colony formation of nup116Δ cells. Expression of constructs with a deletion of the NRM (Nup116-CCΔNRM) in the carboxyl-terminal half of Nup116-C or with only the amino-terminal half (Nup116-CN) present resulted in viability. Expression of GST alone did not inhibit growth of the nup116Δ strain. (C) Immunoblot analysis of the strains shown in B. Lane 1, Nup116-C expression in NUP116 cells (W303α). Lanes 2–8, expression in nup116Δ cells (SWY27). Yeast lysates were separated on a 10.5% SDS-polyacrylamide gel and transferred to nitrocellulose for immunoblotting with an affinity-purified rabbit polyclonal antibody recognizing GST. Molecular mass markers are in kDa.The Integral Membrane Protein Snl1p Is Genetically Linked to Yeast Nuclear Pore Complex FunctionMol Biol Cell. 1998 Feb;9(2):355-373.Figure 2The nup116-C lethal strain exhibits NPC-associated herniations of the nuclear envelope. SWY27 cells harboring the galactose-inducible Nup116-C plasmid (pSW171) were grown in SM-trp 2% raffinose at 23°C and then shifted to SM-trp 2% galactose (A and B) or SM-trp 2% glucose (C) for 15 h. Cells were subsequently processed for thin section electron microscopy. The arrowheads in A and B point to representative nuclear membrane herniations that are attached to NPC-like structures at the nuclear base. The arrows in C indicate wild-type NPCs. Bar, 250 nm.The Integral Membrane Protein Snl1p Is Genetically Linked to Yeast Nuclear Pore Complex FunctionMol Biol Cell. 1998 Feb;9(2):355-373.Figure 3The S. cerevisiae SNL1 gene. (A) A fragment sufficient in high copy for rescuing the nup116-C phenotype was isolated from a yeast genomic library plasmid (pSW807). The library insert contained two complete and two partial ORFs from a region of chromosome IX. The ORF in pSW573 was designated SNL1 (in the yeast genome database as accession number YIL016W). + indicates the plasmid suppressed the nup116-C lethal phenotype, whereas − represents no suppression. The 2μ represents high-copy vector; CEN, low-copy vector. (B) The 159-residue amino acid sequence of the protein encoded by SNL1. A putative transmembrane segment is underlined. (C) hydropathy analysis of Snl1p revealed a span of sequence from amino acids 16 to 35 with significant hydropathic character.The Integral Membrane Protein Snl1p Is Genetically Linked to Yeast Nuclear Pore Complex FunctionMol Biol Cell. 1998 Feb;9(2):355-373.Figure 4Alignment of the Snl1p and Pom152p sequences. An ALIGN analysis between the FL proteins revealed significant homology (). The center line designates the identical (capital letter) and conserved (:, .) residues. The boxed residues in the Snl1p sequence (16–35) and Pom152p (177–195) note the position of the respective predicted transmembrane segments. Pom152p is predicted to be oriented as a type II integral membrane protein (); however, Pom152p topology has not been experimentally determined. Snl1p is positioned with its C-terminal region cytoplasmically exposed (Figure B). If the sequence similarity is significant, this suggests a similar, potential type I orientation for Pom152p.The Integral Membrane Protein Snl1p Is Genetically Linked to Yeast Nuclear Pore Complex FunctionMol Biol Cell. 1998 Feb;9(2):355-373.Figure 5Snl1p is localized to the nuclear envelope/endoplasmic reticulum. snl1Δ cells alone (bottom panels) or harboring a high-copy plasmid expressing a protein A–Snl1p fusion (top panels) and wild-type W303α cells (middle panels) were grown to early log phase in YPD at 30°C before they were fixed in methanol/formaldehyde as described (see MATERIALS AND METHODS). Fixed cells were incubated with rabbit anti-mouse IgG (αIgG) or affinity- purified rabbit polyclonal antibodies raised against Snl1p (αSnl1p) as designated. Binding was detected with an FITC-conjugated goat anti-rabbit IgG. Perinuclear and peripheral staining was observed in wild-type cells, but not in cells lacking Snl1p. The DAPI staining delineating the location of the nucleus is shown on the right. Bar, 2.5 μm.The Integral Membrane Protein Snl1p Is Genetically Linked to Yeast Nuclear Pore Complex FunctionMol Biol Cell. 1998 Feb;9(2):355-373.Figure 6Double immunofluorescence comparison of Snl1p localization to Pom152p and peripheral nucleoporins. Diploid W303 cells were processed for indirect immunofluorescence microscopy as described in MATERIALS AND METHODS. Fixed cells were incubated with affinity-purified rabbit polyclonal anti-Snl1p antibodies (αSnl1p) and with mouse monoclonal anti-Pom152p antibodies (αPom152p, mAb118C3) or mouse monoclonal anti-nucleoporin antibodies (mAb414). The antibodies were detected with Texas red goat anti-rabbit IgG and FITC-labeled goat anti-mouse IgG. Identical fields of cells are shown in A and B. Bar, 3 μm.The Integral Membrane Protein Snl1p Is Genetically Linked to Yeast Nuclear Pore Complex FunctionMol Biol Cell. 1998 Feb;9(2):355-373.Figure 7Immunoblot analysis of yeast subcellular fractionation. Samples of total yeast cells (lane 1), crude cytosol (lane 2), crude nuclei/membranes (lane 3), and sucrose gradient fractions resolving the crude nuclei/membrane fraction (lanes 4–8) were prepared as described in MATERIALS AND METHODS, separated electrophoretically on a 13% SDS-polyacrylamide gel, and transferred to nitrocellulose. The blot was divided in thirds, and the top portion was probed with an affinity-purified antibody recognizing Nup116p, the middle portion with antibody for Kar2p, and the bottom portion with antibody for Snl1p. All three proteins enriched in the crude nuclei/membrane fraction. Loading equivalents represent the number of cell equivalents that were used as starting material to prepare each of the samples.The Integral Membrane Protein Snl1p Is Genetically Linked to Yeast Nuclear Pore Complex FunctionMol Biol Cell. 1998 Feb;9(2):355-373.Figure 8Snl1p is an integral membrane protein, oriented with the C-terminal region accessible to the cytoplasm. (A) Purified nuclear envelopes (NE, lane 1) from W303 diploid nuclei were treated with 0.1 M Na2CO3 (pH 11) and separated into supernatant (S, lane 2) and pellet (P, lane 3) fractions by centrifugation. Samples were separated on a 13% SDS-polyacrylamide gel, transferred to nitrocellulose, and immunoblotted. Strips corresponding to the molecular mass of Nup116p (top) and Snl1p (bottom) were cut from the same blot and probed with the respective antibodies. (B) Cell extracts from a strain expressing an Snl1p–Suc2p fusion protein were either mock treated (0, lane 1) or treated with EndoH (lane 2, 3) as described in MATERIALS AND METHODS (mU = milliunits). The samples were TCA precipitated and resuspended in SDS sample buffer for separation on a 7% SDS-polyacrylamide gel. Immunoblot analysis was performed for Kex2p (top) and Snl1p–Suc2p (bottom). Molecular mass markers are indicated in kDa.The Integral Membrane Protein Snl1p Is Genetically Linked to Yeast Nuclear Pore Complex FunctionMol Biol Cell. 1998 Feb;9(2):355-373.Figure 9Genetic interactions among SNL1, GLE2, NUP116, and NIC96. (A) High-copy SNL1 suppresses the temperature sensitivity of the gle2–1 mutant and the nic96-G3 mutant. The indicated mutant strains were transformed with either pGAL-SNL1 (pSW534), p2μ-SNL1 (pSW807), pCEN-SNL1 (pSW574), or pRS426 (empty 2μ URA3). The strains were streaked to SM-ura 2% galactose to induce overexpression of Snl1p (left) or on SM-ura-leu 2% glucose (right) and grown for 5 d at 37°C. The gle1–4 cells expressing high-copy SNL1 were also inviable when grown at 30°C (lowest nonpermissive growth temperature). The nic96-G3 colonies are white (right), whereas the other strains are red/pink (left and in B). Therefore, different photographic contrasts were used to document the results. (B) Overexpression of SNL1, GLE2, and NIC96 suppresses the nup116-C lethal phenotype. nup116Δ cells alone or harboring pGAL10-GST-nup116-C (pSW171) (nup116-C) were transformed with a CEN pNUP116 plasmid as a control or with the designated 2μ plasmids harboring SNL1, GLE2, NIC96, KAP95, or NPL4. To select for the respective plasmids, the nup116-C cells were grown on SM-trp-ura 2% galactose (left) or SM-trp-leu 2% galactose (right) for 7 d at 23°C. The nup116Δ cells were grown on SM-ura 2% glucose (left) or SM-leu 2% glucose (right) for 7 d at 37°C. Strains harboring a 2μ GLE1 plasmid behaved the same as those with pKAP95 (no colony formation; our unpublished results).The Integral Membrane Protein Snl1p Is Genetically Linked to Yeast Nuclear Pore Complex FunctionMol Biol Cell. 1998 Feb;9(2):355-373.Figure 10The amino-terminal region of Nup116p is required for in vitro interaction with Gle2p. Gle2p-HA was cotranslated in vitro with the indicated Nup116p and Nup145p polypeptides (see Figure A). (A) Autoradiograph of a 7% SDS-polyacrylamide gel with aliquots of the respective cotranslation input fractions used for immunoprecipitation in B. Lane 1 is a translation of Gle2p alone. The position of the Gle2p doublet is noted by the arrow. The upper bands correspond to the respective Nup116p or Nup145p products. (B and C) The 35S-labeled polypeptide mixtures were immunoprecipitated with affinity-purified rabbit polyclonal anti-GLFG antibody (B) or mAb12CA5 (anti-HA/Gle2p, C) and protein A– Sepharose beads. The bound fraction was eluted by boiling in SDS sample buffer and separated by electrophoresis on either a 7% (B) or 13% (C) SDS-polyacrylamide gel. 35S-labeled proteins were detected by autoradiography. Molecular mass markers are noted in kDa.The Integral Membrane Protein Snl1p Is Genetically Linked to Yeast Nuclear Pore Complex FunctionMol Biol Cell. 1998 Feb;9(2):355-373.Figure 11Schematic diagram of molecular links between the functions of SNL1, GLE2, NUP116, NUP100, NIC96, and POM152. The dashed lines indicate a synthetic lethal phenotype between mutant alleles, the dashed lines with arrowheads indicate high-copy suppression of a mutant allele by a wild-type gene (with the orientation pointing toward the conditional phenotype of the gene being suppressed), and the solid line indicates direct in vitro protein–protein interactions.The Integral Membrane Protein Snl1p Is Genetically Linked to Yeast Nuclear Pore Complex FunctionMol Biol Cell. 1998 Feb;9(2):355-373.Publication type, MeSH terms, Substances, Grant supportPublication typeResearch Support, U.S. Gov't, P.H.S.MeSH termsAmino Acid SequenceCloning, MolecularEndoplasmic Reticulum/chemistryFungal Proteins/metabolismGenes, Fungal/geneticsGenes, Lethal/geneticsGenes, Suppressor/geneticsMembrane Glycoproteins/analysisMembrane Glycoproteins/geneticsMembrane Proteins/analysisMembrane Proteins/genetics*Membrane Proteins/metabolismMembrane Proteins/physiology*Molecular ChaperonesMolecular Sequence DataNuclear Envelope/chemistryNuclear Envelope/ultrastructureNuclear PoreNuclear Pore Complex Proteins*Nuclear Proteins/analysisNuclear Proteins/genetics*Nuclear Proteins/metabolismPhenotypeSaccharomyces cerevisiae/cytology*Saccharomyces cerevisiae/geneticsSaccharomyces cerevisiae Proteins*Sequence AlignmentSequence Homology, Amino AcidSubstancesFungal ProteinsMembrane GlycoproteinsMembrane ProteinsMolecular ChaperonesNIC96 protein, S cerevisiaeNUP116 protein, S cerevisiaeNuclear Pore Complex ProteinsNuclear ProteinsPOM152 protein, S cerevisiaeSNL1 protein, S cerevisiaeSaccharomyces cerevisiae ProteinsGrant supportR01 GM051219/GM/NIGMS NIH HHS/United StatesR37 GM051219/GM/NIGMS NIH HHS/United StatesGM-51219/GM/NIGMS NIH HHS/United StatesLinkOut - more resourcesFull Text SourcesHighWireEurope PubMed CentralPubMed CentralPubMed Central CanadaOther Literature SourcesCOS Scholar UniverseMolecular Biology DatabasesGene Ontology annotations from this paper - Gene OntologySaccharomyces Genome DatabasePubMed Commons home