The NUP1 gene encodes an essential component of the yeast nuclear pore complex

Monoclonal antibodies generated against a family of related nuclear pore complex proteins (nucleoporins) from rat liver nuclei cross-react with several proteins in the yeast S. cerevisiae and show punctate nuclear envelope staining similar to the pattern seen in mammalian cells. We have cloned a gene encoding one of these proteins (NUP1) and have confirmed the localization of the NUP1 protein to the pore complex by immunofluorescence, using an epitope-tagged construct to differentiate it from other members of this family. The NUP1 protein is essential for cell viability, and overexpression from the yeast GAL10 promoter prevents further cell growth. The central domain of NUP1 consists of a series of degenerate repeats similar to those found in the nucleoskeletal protein NSP1, a protein that cross-reacts with monoclonal antibodies against NUP1. We propose that the repetitive domain is a feature common to the nucleoporins.     

鼠肝炎冠状病毒非结构蛋白1及其突变体的原核表达

目的:构建鼠肝炎冠状病毒(MHV)非结构蛋白1(NSP1)及其突变体(NSP1 mu)的原核重组表达质粒,在大肠杆菌中分别融合表达重组NSP1及NSP1 mu。方法:以现有质粒载体为模板,扩增编码NSP1及NSP1 mu的基因片段,并克隆至pMD18-T克隆载体;菌落PCR鉴定阳性克隆并测序分析;将阳性克隆的目的片段亚克隆至表达载体pET-28a,并转化大肠杆菌TOP10感受态细胞,PCR和双酶切鉴定转化菌落;将阳性质粒转化大肠杆菌BL21(DE3)感受态细胞并加入IPTG诱导表达,SDS-PAGE和免疫印迹分析目的蛋白的表达。结果:PCR扩增得到表达NSP1及NSP1 mu的特异片段,并克隆到pMD18-T载体,测序结果正确无误;构建了NSP1和NSP1 mu的重组表达质粒,并在大肠杆菌BL21(DE3)中分别融合表达了重组NSP1及NSP1 mu,表达的目的蛋白均能与His单克隆抗体特异结合;用Ni-NTA琼脂糖试剂盒纯化重组蛋白,获得可溶性的NSP1及NSP1 mu,相对分子质量分别为27×103和28×103。结论:在大肠杆菌中分别表达并纯化获得了大量可溶性重组NSP1及NSP1 mu。     

Purification of NSP1 reveals complex formation with 'GLFG' nucleoporins and a novel nuclear pore protein NIC96.

The essential C-terminal domain of NSP1 mediates assembly into the nuclear pore complex (NPC). To identify components which interact physically with this yeast nucleoporin, the tagged C-terminal domain of NSP1 (ProtA-NSP1) was isolated by affinity chromatography under non-denaturing conditions. The purified complex contains ProtA-NSP1, two previously identified 'GLFG' nucleoporins, NUP49 (NSP49) and p54 and a novel protein designated NIC96 (for Nucleoporin-Interacting Component of 96 kDa). Conversely, affinity purification of tagged NSP49 enriches for NSP1, the p54 and the NIC96 component. The NIC96 gene was cloned; it encodes a novel 839 amino acid protein essential for cell growth. By immunofluorescence, protein A-tagged NIC96 exhibits a punctate nuclear membrane staining indicative of nuclear pore location. Therefore, affinity purification of tagged nucleoporins has allowed the definition of a subcomplex of the NPC and analysis of physical interactions between nuclear pore proteins.     

Energy- and temperature-dependent transport of integral proteins to the inner nuclear membrane via the nuclear pore

Resident integral proteins of the inner nuclear membrane (INM) are synthesized as membrane-integrated proteins on the peripheral endoplasmic reticulum (ER) and are transported to the INM throughout interphase using an unknown trafficking mechanism. To study this transport, we developed a live cell assay that measures the movement of transmembrane reporters from the ER to the INM by rapamycin-mediated trapping at the nuclear lamina. Reporter constructs with small (<30 kD) cytosolic and lumenal domains rapidly accumulated at the INM. However, increasing the size of either domain by 47 kD strongly inhibited movement. Reduced temperature and ATP depletion also inhibited movement, which is characteristic of membrane fusion mechanisms, but pharmacological inhibition of vesicular trafficking had no effect. Because reporter accumulation at the INM was inhibited by antibodies to the nuclear pore membrane protein gp210, our results support a model wherein transport of integral proteins to the INM involves lateral diffusion in the lipid bilayer around the nuclear pore membrane, coupled with active restructuring of the nuclear pore complex.     

Reconstitution of nuclear protein transport with semi-intact yeast cells

We have developed an in vitro nuclear protein import reaction from semi- intact yeast cells. The reaction uses cells that have been permeabilized by freeze-thaw after spheroplast formation. Electron microscopic analysis and antibody-binding experiments show that the nuclear envelope remains intact but the plasma membrane is perforated. In the presence of ATP and cytosol derived from yeast or mammalian cells, a protein containing the nuclear localization sequence (NLS) of SV40 large T-antigen is transported into the nucleus. Proteins with mutant NLSs are not imported. In the absence of cytosol, binding of NLS- containing proteins occurs at the nuclear envelope. N-ethylmaleimide treatment of the cytosol as well as antibodies to the nuclear pore protein Nsp1 inhibit import but not binding to the nuclear envelope. Yeast mutants defective in nuclear protein transport were tested in the in vitro import reaction. Semi-intact cells from temperature-sensitive nsp1 mutants failed to import but some binding to the nuclear envelope was observed. On the other hand, no binding and thus no import into nuclei was observed in semi-intact nsp49 cells which are mutated in another nuclear pore protein. Np13 mutants, which are defective for nuclear protein import in vivo, were also deficient in the binding step under the in vitro conditions. Thus, the transport defect in these mutants is at the level of the nucleus and the point at which nuclear transport is blocked can be defined.     

Human nucleoporin p62 and the essential yeast nuclear pore protein NSP1 show sequence homology and a similar domain organization

NSP1 is an essential nuclear pore protein in yeast. We observed that anti-NSP1 antibodies label mammalian nuclear pore complexes and recognize nucleoporin p62. Also peptide antibodies raised against the NSP1 carboxy-terminal end cross-react with p62, a conserved component of the nuclear pore complex in higher eukaryotes. To further analyze the structural and functional similarity between NSP1 and mammalian nucleoporins, we cloned and sequenced nucleoporin p62 from a HeLa cDNA library. Human p62 consists of a carboxy-terminal domain homologous to the essential yeast NSP1 carboxy-terminal domain and an amino-terminal half resembling the repetitive middle domain of NSP1. The full-length p62 and a fusion protein consisting of cytosolic mouse dihydrofolate reductase (DHFR) and the p62 carboxy-terminal domain were expressed in transfected HeLa cells. Only overexpressed full-length p62, but not the DHFR-C-p62 fusion protein, binds wheat germ agglutinin (WGA). This suggests that modification by N-acetylglucosamine is mainly restricted to the repetitive amino-terminal half of p62 and implies a role of this type of repetitive sequences in nuclear transport. In the transfected HeLa cells, the DHFR-C-p62 fusion protein forms patchy aggregates that accumulate at the nuclear periphery but are also scattered through the cytoplasm. It is suggested that nucleoporin p62 may be targeted and anchored to the pore complex via its carboxy-terminal domain which reveals a hydrophobic heptad repeat organization similar to that found in lamins and other intermediate filament proteins.     

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

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.     

NSP1: a yeast nuclear envelope protein localized at the nuclear pores exerts its essential function by its carboxy-terminal domain

NSP1 is located at the nuclear periphery in yeast and is essential for cell growth. Employing immunoelectron microscopy on yeast cells, we show that NSP1 is located at the nuclear pores. The molecular analysis of the NSP1 protein points to a two domain model: a nonessential domain (the first 603 amino acids) composed of repetitive sequences common to other nuclear proteins and an essential, carboxy-terminal domain (residues 604-823) mediating the vital function of NSP1. The NSP1 carboxy-terminal domain, which shows a heptad repeat organization, affected the correct location of two nuclear proteins: site-specific amino acid substitutions within a predicted alpha-helical structure of this domain caused a temperature-sensitive growth arrest at 37 degrees C and the appearance of NSP1 and NOP1, a nucleolar protein, in the cytosol.     

A new subclass of nucleoporins that functionally interact with nuclear pore protein NSP1.

NSP1 is a nuclear pore protein (nucleoporin) essential for cell growth. To identify the components that functionally interact with NSP1 in the living cell, we developed a genetic screen for mutants that are lethal in a genetic background of mutated, but not wild type NSP1. Fourteen synthetic lethal mutants were obtained, belonging to at least four different complementation groups. The genes of two complementation groups, NSP116 and NSP49, were cloned. Like the previously described nucleoporins, these genes encode proteins with many repeat sequences. NSP116 and NSP49, however, contain a new repetitive sequence motif 'GLFG', which classifies them as a subclass of nucleoporins. NSP116 and NSP49, tagged with the IgG binding domain of protein A and expressed in yeast, are located at the nuclear envelope. These data provide in vivo evidence that distinct subclasses of nucleoporins physically interact or share overlapping function in nuclear pore complexes.     

Targeting of a cytosolic protein to the nuclear periphery

The yeast nuclear envelope protein NSP1 is located at the nuclear pores and mediates its essential function via the carboxy-terminal domain. The passenger protein, cytosolic dihydrofolate reductase from mouse, was fused to the 220 residue long NSP1 carboxy-terminal domain. When expressed in yeast, this chimeric protein was tightly associated with nuclear structures and was localized at the nuclear periphery very similar to authentic NSP1. Furthermore, the DHFR-C-NSP1 fusion protein was able to complement a yeast mutant lacking a functional NSP1 gene showing that DHFR-C-NSP1 fulfils the same basic function as compared to the endogenous NSP1 protein. These data also show that the NSP1 protein is composed of separate functional moieties: a carboxy-terminal domain that is sufficient to mediate the association with the nuclear periphery and an amino-terminal and middle repetitive domain with an as yet unknown function. It is suggested that heptad repeats found in the NSP1 carboxy-terminal domain, which are similar to those found in intermediate filament proteins, are crucial for mediating the association with the nuclear pores.     

Characterization of nuclear polyadenylated RNA-binding proteins in Saccharomyces cerevisiae

To study the functions of heterogeneous nuclear ribonucleoproteins (hnRNPs), we have characterized nuclear polyadenylated RNA-binding (Nab) proteins from Saccharomyces cerevisiae. Nab1p, Nab2p, and Nab3p were isolated by a method which uses UV light to cross-link proteins directly bound to poly(A)+ RNA in vivo. We have previously characterized Nab2p, and demonstrated that it is structurally related to human hnRNPs. Here we report that Nab1p is identical to the Np13p/Nop3p protein recently implicated in both nucleocytoplasmic protein shuttling and pre-rRNA processing, and characterize a new nuclear polyadenylated RNA-binding protein, Nab3p. The intranuclear distributions of the Nab proteins were analyzed by three-dimensional immunofluorescence optical microscopy. All three Nab proteins are predominantly localized within the nucleoplasm in a pattern similar to the distribution of hnRNPs in human cells. The NAB3 gene is essential for cell viability and encodes an acidic ribonucleoprotein. Loss of Nab3p by growth of a GAL::nab3 mutant strain in glucose results in a decrease in the amount of mature ACT1, CYH2, and TPI1 mRNAs, a concomitant accumulation of unspliced ACT1 pre-mRNA, and an increase in the ratio of unspliced CYH2 pre-mRNA to mRNA. These results suggest that the Nab proteins may be required for packaging pre-mRNAs into ribonucleoprotein structures amenable to efficient nuclear RNA processing.     

NUP2, a novel yeast nucleoporin, has functional overlap with other proteins of the nuclear pore complex.

We have isolated a new gene, NUP2, that encodes a constituent of the yeast-nuclear pore complex (NPC). The NUP2 protein sequence shares a central repetitive domain with NSP1 and NUP1, the two previously characterized yeast nucleoporins. Like NUP1 and NSP1, NUP2 localizes to discrete spots in the nuclear envelope, as determined by indirect immunofluorescence. Although the sequence similarity among these three nucleoporins suggests that they have a similar role in the nuclear pore complex, NUP2, in contrast to NSP1 and NUP1, is not required for growth. Some combinations of mutant alleles of NUP1, NSP1, and NUP2 display "synthetic lethal" relationships that provide evidence for functional interaction between these NPC components. This genetic evidence of overlapping function suggests that the nucleoporins act in concert, perhaps participating in the same step of the recognition or transit of macromolecules through the NPC.     

Rea1, a dynein-related nuclear AAA-ATPase, is involved in late rRNA processing and nuclear export of 60 S subunits

Rea1, the largest predicted protein in the yeast genome, is a member of the AAA(+) family of ATPases and is associated with pre-60 S ribosomes. Here we report that Rea1 is required for maturation and nuclear export of the pre-60 S subunit. Rea1 exhibits a predominantly nucleoplasmic localization and is present in a late pre-60 S particle together with members of the Rix1 complex. To study the role of Rea1 in ribosome biogenesis, we generated a repressible GAL::REA1 strain and temperature-sensitive rea1 alleles. In vivo depletion of Rea1 results in the significant reduction of mature 60 S subunits concomitant with defects in pre-rRNA processing and late pre-60 S ribosome stability following ITS2 cleavage and prior to the generation of mature 5.8 S rRNA. Strains depleted of the components of the Rix1 complex (Rix1, Ipi1, and Ipi3) showed similar defects. Using an in vivo 60 S subunit export assay, a strong accumulation of the large subunit reporter Rpl25-GFP (green fluorescent protein) in the nucleus and at the nuclear periphery was seen in rea1 mutants at restrictive conditions.     

A novel nuclear pore protein Nup133p with distinct roles in poly(A)+ RNA transport and nuclear pore distribution.

Temperature-sensitive nucleoporin nup49-316 mutant cells accumulate poly(A)+ RNA inside the nucleus when shifted to restrictive temperature. We performed a synthetic lethal screen with this mutant allele to identify further components of the mRNA export machinery. A synthetic lethal mutant slv21 was isolated, which exhibited a ts phenotype and showed nuclear accumulation of poly(A)+ RNA at 37 degrees C. The wild-type gene complementing slv21 was cloned and sequenced. It encodes a novel protein Nup133p which is located at the nuclear pore complex. NUP133 is not an essential gene, but cells in which NUP133 is disrupted grow slowly at permissive temperatures and stop growing at 37 degrees C. Concomitant with the growth inhibition, nup133- cells accumulate poly(A)+ RNA inside the nucleus whereas nuclear import of a karyophilic reporter protein is not altered. Strikingly, nup133- cells display extensive clustering of nuclear pore complexes at a few sites on the nuclear envelope. However, the nuclear pore clustering phenotype and intranuclear accumulation of poly(A)+ RNA are not obligatorily linked, since an amino-terminally truncated Nup133p allows normal poly(A)+ RNA export, but does not complement the clustering phenotype of nup133- cells.