Quantitative Analysis of Membrane Protein Transport Across the Nuclear Pore Complex

Nuclear transport of the Saccharomyces cerevisiae membrane proteins Src1/Heh1 and Heh2 across the NPC is facilitated by a long intrinsically disordered linker between the nuclear localization signal (NLS) and the transmembrane domain. The import of reporter proteins derived from Heh2 is dependent on the FG‐Nups in the central channel, and the linker can position the transport factor‐bound NLS in the vicinity of the FG‐Nups in the central channel, while the transmembrane segment resides in the pore membrane. Here, we present a quantitative analysis of karyopherin‐mediated import and passive efflux of reporter proteins derived from Heh2, including data on the mobility of the reporter proteins in different membrane compartments. We show that membrane proteins with extralumenal domains up to 174 kDa, terminal to the linker and NLS, passively leak out of the nucleus via the NPC, albeit at a slow rate. We propose that also during passive efflux, the unfolded linker facilitates the passage of extralumenal domains through the central channel of the NPC .     

Localization of Pom121 to the inner nuclear membrane is required for an early step of interphase nuclear pore complex assembly

The nuclear pore complex (NPC) is a large protein assembly that mediates molecular trafficking between the cytoplasm and the nucleus. NPCs assemble twice during the cell cycle in metazoans: postmitosis and during interphase. In this study, using small interfering RNA (siRNA) in conjunction with a cell fusion-based NPC assembly assay, we demonstrated that pore membrane protein (Pom)121, a vertebrate-specific integral membrane nucleoporin, is indispensable for an early step in interphase NPC assembly. Functional domain analysis of Pom121 showed that its nuclear localization signals, which bind to importin β via importin α and likely function with RanGTP, play an essential role in targeting Pom121 to the interphase NPC. Furthermore, a region of Pom121 that interacts with the inner nuclear membrane (INM) and lamin B receptor was found to be crucial for its NPC targeting. Based on these findings and on evidence that Pom121 localizes at the INM in the absence of a complete NPC structure, we propose that the nuclear migration of Pom121 and its subsequent interaction with INM proteins are required to initiate interphase NPC assembly. Our data also suggest, for the first time, the importance of the INM as a seeding site for "prepores" during interphase NPC assembly.     

Lumenal interactions in nuclear pore complex assembly and stability

Nuclear pore complexes (NPCs) provide a gateway for the selective transport of macromolecules across the nuclear envelope (NE). Although we have a solid understanding of NPC composition and structure, we do not have a clear grasp of the mechanism of NPC assembly. Here, we demonstrate specific defects in nucleoporin distribution in strains lacking Heh1p and Heh2p-two conserved members of the LEM (Lap2, emerin, MAN1) family of integral inner nuclear membrane proteins. These effects on nucleoporin localization are likely of functional importance as we have defined specific genetic interaction networks between HEH1 and HEH2, and genes encoding nucleoporins in the membrane, inner, and outer ring complexes of the NPC. Interestingly, expression of a domain of Heh1p that resides in the NE lumen is sufficient to suppress both the nucleoporin mislocalization and growth defects in heh1Δpom34Δ strains. We further demonstrate a specific physical interaction between the Heh1p lumenal domain and the massive cadherin-like lumenal domain of the membrane nucleoporin Pom152p. These findings support a role for Heh1p in the assembly or stability of the NPC, potentially through the formation of a lumenal bridge with Pom152p.     

核定位信号及其分析策略

真核细胞核膜上的核孔复合体 (nuclear pore complex, NPC) 是细胞核内外进行物质交换的主要通道, 分子量较小的化合物可自由通过NPC或采取被动扩散的方式进入细胞核, 而分子量为50 kD以上的蛋白质则只能通过主动转运进入细胞核. 以这种方式进入细胞核的 蛋白质必须在其氨基酸序列上拥有特殊的核定位信号(nuclear localization signal, NLS)以被相应的核转运蛋白(karyopherins) 识别. 核定位信号具有多样性, 包括经典核定位信号(classical NLS,cNLS), 内输蛋白β2识别的核定位信号（又称PY模体-NLS）和其它类型的NLS. 每一类NLS具有相似的特征, 但并不具有完全保守的氨基酸组成. 不同的NLS, 往往对应着各不相同的核输入机制. 而对同一蛋白质来说, 也可能同时拥有几个功能性的NLS. 研究核定位信号一方面可以帮助揭示新的大分子物质核转运机制, 另一方面也有助于发现一些蛋白质的新功能. 本文对常见NLS的分类进行了总结, 并介绍了两种常用的NLS预测软件及鉴定NLS的一般策略.     

The role of the integral membrane nucleoporins Ndc1p and Pom152p in nuclear pore complex assembly and function

The nuclear pore complex (NPC) is a large channel that spans the two lipid bilayers of the nuclear envelope and mediates transport events between the cytoplasm and the nucleus. Only a few NPC components are transmembrane proteins, and the role of these proteins in NPC function and assembly remains poorly understood. We investigate the function of the three integral membrane nucleoporins, which are Ndc1p, Pom152p, and Pom34p, in NPC assembly and transport in Saccharomyces cerevisiae. We find that Ndc1p is important for the correct localization of nuclear transport cargoes and of components of the NPC. However, the role of Ndc1p in NPC assembly is partially redundant with Pom152p, as cells lacking both of these proteins show enhanced NPC disruption. Electron microscopy studies reveal that the absence of Ndc1p and Pom152p results in aberrant pores that have enlarged diameters and lack proteinaceous material, leading to an increased diffusion between the cytoplasm and the nucleus.     

Pom121 links two essential subcomplexes of the nuclear pore complex core to the membrane

Nuclear pore complexes (NPCs) control the movement of molecules across the nuclear envelope (NE). We investigated the molecular interactions that exist at the interface between the NPC scaffold and the pore membrane. We show that key players mediating these interactions in mammalian cells are the nucleoporins Nup155 and Nup160. Nup155 depletion massively alters NE structure, causing a dramatic decrease in NPC numbers and the improper targeting of membrane proteins to the inner nuclear membrane. The role of Nup155 in assembly is likely closely linked to events at the membrane as we show that Nup155 interacts with pore membrane proteins Pom121 and NDC1. Furthermore, we demonstrate that the N terminus of Pom121 directly binds the β-propeller regions of Nup155 and Nup160. We propose a model in which the interactions of Pom121 with Nup155 and Nup160 are predicted to assist in the formation of the nuclear pore and the anchoring of the NPC to the pore membrane.     

Structural basis for the specificity of bipartite nuclear localization sequence binding by importin-alpha

Importin-alpha is the nuclear import receptor that recognizes cargo proteins carrying conventional basic monopartite and bipartite nuclear localization sequences (NLSs) and facilitates their transport into the nucleus. Bipartite NLSs contain two clusters of basic residues, connected by linkers of variable lengths. To determine the structural basis of the recognition of diverse bipartite NLSs by mammalian importin-alpha, we co-crystallized a non-autoinhibited mouse receptor protein with peptides corresponding to the NLSs from human retinoblastoma protein and Xenopus laevis phosphoprotein N1N2, containing diverse sequences and lengths of the linker. We show that the basic clusters interact analogously in both NLSs, but the linker sequences adopt different conformations, whereas both make specific contacts with the receptor. The available data allow us to draw general conclusions about the specificity of NLS binding by importin-alpha and facilitate an improved definition of the consensus sequence of a conventional basic/bipartite NLS (KRX10-12KRRK) that can be used to identify novel nuclear proteins.     

NLS-mediated NPC functions of the nucleoporin Pom121

RanGTP mediates nuclear import and mitotic spindle assembly by dissociating import receptors from nuclear localization signal (NLS) bearing proteins. We investigated the interplay between import receptors and the transmembrane nucleoporin Pom121. We found that Pom121 interacts with importin α/β and a group of nucleoporins in an NLS-dependent manner. In vivo, replacement of Pom121 with an NLS mutant version resulted in defective nuclear transport, induction of aberrant cytoplasmic membrane stacks and decreased cell viability. We propose that the NLS sites of Pom121 affect its function in NPC assembly both by influencing nucleoporin interactions and pore membrane structure.

Structured summary

MINT-7951230: pom121 (uniprotkb:Q5EWX9) physically interacts (MI:0914) with nup155 (uniprotkb:O75694), Nup133 (uniprotkb:Q8WUM0) and Importin beta (uniprotkb:Q14974) by pull down (MI:0096)MINT-7951210: pom121 (uniprotkb:Q5EWX9) physically interacts (MI:0915) with Importin alpha (uniprotkb:P52170) and Importin beta (uniprotkb:P52297) by pull down (MI:0096)MINT-7951183: pom121 (uniprotkb:Q5EWX9) physically interacts (MI:0914) with nup155 (uniprotkb:Q7ZWL0), nup160 (uniprotkb:P83722), nup205 (uniprotkb:Q642R6), nup93 (uniprotkb:Q7ZX96), Importin beta (uniprotkb:P52297) and nup62 (uniprotkb:Q91349) by pull down (MI:0096)MINT-7951416: pom121 (uniprotkb:Q5EWX9) physically interacts (MI:0914) with nup155 (uniprotkb:Q7ZWL0), nup93 (uniprotkb:Q7ZX96) and Importin beta (uniprotkb:P52297) by pull down (MI:0096)MINT-7951276: pom121 (uniprotkb:Q5EWX9) physically interacts (MI:0914) with nup155 (uniprotkb:Q7ZWL0), nup205 (uniprotkb:Q642R6), nup93 (uniprotkb:Q7ZX96), Importin beta (uniprotkb:P52297) and nup62 (uniprotkb:Q91349) by pull down (MI:0096)MINT-7951306, MINT-7951362: pom121 (uniprotkb:Q5EWX9) physically interacts (MI:0914) with nup155 (uniprotkb:Q7ZWL0), nup160 (uniprotkb:P83722), nup93 (uniprotkb:Q7ZX96), Importin beta (uniprotkb:P52297) and nup62 (uniprotkb:Q91349) by pull down (MI:0096)     

Two distinct human POM121 genes: requirement for the formation of nuclear pore complexes

Pom121 is one of the integral membrane components of the nuclear pore complex (NPC) in vertebrate cells. Unlike rodent cells carrying a single POM121 gene, human cells possess multiple POM121 gene loci on chromosome 7q11.23, as a consequence of complex segmental-duplications in this region during human evolution. In HeLa cells, two "full-length" Pom121 are transcribed and translated by two distinct genetic loci. RNAi experiments showed that efficient depletion of both Pom121 proteins significantly reduces assembled NPCs on nuclear envelope. Pom121-depletion also induced clustering of NPCs, indicating its role on maintenance of NPC structure/organization.     

细胞因子和生长因子信号转导途径中信号蛋白分子的核转运机制

信号蛋白分子的入核及出核转运是细胞因子和生长因子信号转导途径中的重要环节.核定位序列（NLS）是信号蛋白分子上与入核转运相关的氨基酸序列.核孔复合物（NPC）、核转运蛋白importin和能量供应体Ran/TC4在入核转运过程中也发挥了重要作用.另外,很多细胞因子和生长因子或其受体上所含有的NLS序列也具有核定位功能,并可能通过“伴侣机制”参与其他信号蛋白分子的入核转运.     

ER membrane–bending proteins are necessary for de novo nuclear pore formation

Nucleocytoplasmic transport occurs exclusively through nuclear pore complexes (NPCs) embedded in pores formed by inner and outer nuclear membrane fusion. The mechanism for de novo pore and NPC biogenesis remains unclear. Reticulons (RTNs) and Yop1/DP1 are conserved membrane protein families required to form and maintain the tubular endoplasmic reticulum (ER) and the postmitotic nuclear envelope. In this study, we report that members of the RTN and Yop1/DP1 families are required for nuclear pore formation. Analysis of Saccharomyces cerevisiae prp20-G282S and nup133Δ NPC assembly mutants revealed perturbations in Rtn1–green fluorescent protein (GFP) and Yop1-GFP ER distribution and colocalization to NPC clusters. Combined deletion of RTN1 and YOP1 resulted in NPC clustering, nuclear import defects, and synthetic lethality with the additional absence of Pom34, Pom152, and Nup84 subcomplex members. We tested for a direct role in NPC biogenesis using Xenopus laevis in vitro assays and found that anti-Rtn4a antibodies specifically inhibited de novo nuclear pore formation. We hypothesize that these ER membrane–bending proteins mediate early NPC assembly steps.     

Finding nuclear localization signals

A variety of nuclear localization signals (NLSs) are experimentally known although only one motif was available for database searches through PROSITE. We initially collected a set of 91 experimentally verified NLSs from the literature. Through iterated ‘in silico mutagenesis’ we then extended the set to 214 potential NLSs. This final set matched in 43% of all known nuclear proteins and in no known non-nuclear protein. We estimated that >17% of all eukaryotic proteins may be imported into the nucleus. Finally, we found an overlap between the NLS and DNA-binding region for 90% of the proteins for which both the NLS and DNA-binding regions were known. Thus, evolution seemed to have used part of the existing DNA-binding mechanism when compartmentalizing DNA-binding proteins into the nucleus. However, only 56 of our 214 NLS motifs overlapped with DNA-binding regions. These 56 NLSs enabled a de novo prediction of partial DNA-binding regions for ~800 proteins in human, fly, worm and yeast.     

Pom33, a novel transmembrane nucleoporin required for proper nuclear pore complex distribution

The biogenesis of nuclear pore complexes (NPCs) represents a paradigm for the assembly of high-complexity macromolecular structures. So far, only three integral pore membrane proteins are known to function redundantly in NPC anchoring within the nuclear envelope. Here, we describe the identification and functional characterization of Pom33, a novel transmembrane protein dynamically associated with budding yeast NPCs. Pom33 becomes critical for yeast viability in the absence of a functional Nup84 complex or Ndc1 interaction network, which are two core NPC subcomplexes, and associates with the reticulon Rtn1. Moreover, POM33 loss of function impairs NPC distribution, a readout for a subset of genes required for pore biogenesis, including members of the Nup84 complex and RTN1. Consistently, we show that Pom33 is required for normal NPC density in the daughter nucleus and for proper NPC biogenesis and/or stability in the absence of Nup170. We hypothesize that, by modifying or stabilizing the nuclear envelope–NPC interface, Pom33 may contribute to proper distribution and/or efficient assembly of nuclear pores.     

Functional identification of multiple nucleocytoplasmic trafficking signals in the broad-spectrum resistance protein RPW8.2

Nuclear localization signals (NLSs) and nuclear export signals (NESs) are important intramolecular regulatory elements for protein nucleocytoplasmic trafficking. This regulation confers spatial specificity to signal initiation and transduction in eukaryotic cells and thus is fundamental to the viability of all eukaryotic organisms. Here, we developed a simple and rapid method in which activity of putative NLSs or NESs was reported by subcellular localization of two tandem fluorescent proteins in fusion with the respective NLSs or NESs after agroinfiltration-mediated transient expression in leaves of Nicotiana benthamiana (Nb). We further demonstrated that the predicted NES from amino acid residue (aa) 9 to 22 and the NLS from aa91 to 101 in the broad-spectrum disease resistance protein RPW8.2 possess nuclear export and import activity, respectively. Additionally, by testing overlapping fragments covering the full length of RPW8.2, we identified another NLS from aa65 to 74 with strong nuclear import activity and two tandem non-canonical NESs in the C-terminus with strong nuclear export activity. Taken together, our results demonstrated the utility of a simple method to evaluate potential NLSs and NESs in plant cells and suggested that RPW8.2 may be subject to opposing nucleocytoplasmic trafficking forces for its subcellular localization and functional execution.     

Cooperative nuclear localization sequences lend a novel role to the N-terminal region of MSH6

Human mismatch repair proteins MSH2-MSH6 play an essential role in maintaining genetic stability and preventing disease. While protein functions have been extensively studied, the substantial amino-terminal region (NTR*) of MSH6 that is unique to eukaryotic proteins, has mostly evaded functional characterization. We demonstrate that a cluster of three nuclear localization signals (NLS) in the NTR direct nuclear import. Individual NLSs are capable of partially directing cytoplasmic protein into the nucleus; however only cooperative effects between all three NLSs efficiently transport MSH6 into the nucleus. In striking contrast to yeast and previous assumptions on required heterodimerization, human MSH6 does not determine localization of its heterodimeric partner, MSH2. A cancer-derived mutation localized between two of the three NLS significantly decreases nuclear localization of MSH6, suggesting altered protein localization can contribute to carcinogenesis. These results clarify the pending speculations on the functional role of the NTR in human MSH6 and identify a novel, cooperative nuclear localization signal.     

Expanding the Definition of the Classical Bipartite Nuclear Localization Signal

Nuclear localization signals (NLSs) are amino acid sequences that target cargo proteins into the nucleus. Rigorous characterization of NLS motifs is essential to understanding and predicting pathways for nuclear import. The best‐characterized NLS is the classical NLS (cNLS), which is recognized by the cNLS receptor, importin‐α. cNLSs are conventionally defined as having one (monopartite) or two clusters of basic amino acids separated by a 9‐12 aa linker (bipartite). Motivated by the finding that Ty1 integrase, which contains an unconventional putative bipartite cNLS with a 29 aa linker, exploits the classical nuclear import machinery, we assessed the functional boundaries for linker length within a bipartite cNLS. We confirmed that the integrase cNLS is a bona fide bipartite cNLS, then carried out a systematic analysis of linker length in an obligate bipartite cNLS cargo, which revealed that some linkers longer than conventionally defined can function in nuclear import. Linker function is dependent on the sequence and likely the inherent flexibility of the linker. Subsequently, we interrogated the Saccharomyces cerevisiae proteome to identify cellular proteins containing putative long bipartite cNLSs. We experimentally confirmed that Rrp4 contains a bipartite cNLS with a 25 aa linker. Our studies show that the traditional definition of bipartite cNLSs is too restrictive and linker length can vary depending on amino acid composition.     

The homeodomain of Nkx2.2 carries two cooperatively acting nuclear localization signals

NK-2 family members of homeodomain proteins have been identified as important regulators of growth and development in the ventral forebrain, heart, lung, and thyroid. In addition, Nk2.2 expression has been detected in the pancreas, where it is vital for the final differentiation of beta-cells. In our present paper, we have analyzed the domains necessary for nuclear transport of Nkx2.2. With the help of deletion mutants we identified two separate nuclear localization signals (NLS). Interestingly, both NLSs are situated in the homeodomain. They belong to the monopartite class of NLS; the proximal NLS has the sequence KKRKRR and lies at the very N-terminus of the homeodomain, while the more distal NLS RYKMKRAR is at the homeodomain C-terminus. Each NLS per se is sufficient for nuclear transport of Nkx2.2 into the nucleus, although inefficiently. Both identified NLSs act cooperatively in mediating complete nuclear transport of Nkx2.2.