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 .     

Flexible and Extended Linker Domains Support Efficient Targeting of Heh2 to the Inner Nuclear Membrane

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The Inner Nuclear Membrane Protein Bqt4 in Fission Yeast Contains a DNA-Binding Domain Essential for Telomere Association with the Nuclear Envelope

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核孔复合体的动态组装

核孔是介导所有大分子入核出核的唯一通道。在整个生命活动中,核孔复合体的组成蛋白总是处于动态变化中。核孔复合体的动态组装改变了核质转运状态,并最终改变了细胞的功能。     

Mechanism and a Peptide Motif for Targeting Peripheral Proteins to the Yeast Inner Nuclear Membrane

Trm1 is a tRNA specific m₂²G methyltransferase shared by nuclei and mitochondria in Saccharomyces cerevisiae . In nuclei, Trm1 is peripherally associated with the inner nuclear membrane (INM). We investigated the mechanism delivering/tethering Trm1 to the INM. Analyses of mutations of the Ran pathway and nuclear pore components showed that Trm1 accesses the nucleoplasm via the classical nuclear import pathway. We identified a Trm1 cis-acting sequence sufficient to target passenger proteins to the INM. Detailed mutagenesis of this region uncovered specific amino acids necessary for authentic Trm1 to locate at the INM. The INM information is contained within a sequence of less than 20 amino acids, defining the first motif for addressing a peripheral protein to this important subnuclear location. The combined studies provide a multi-step process to direct Trm1 to the INM: (i) translation in the cytoplasm; (ii) Ran-dependent import into the nucleoplasm; and (iii) redistribution from the nucleoplasm to the INM via the INM motif. Furthermore, we demonstrate that the Trm1 mitochondrial targeting and nuclear localization signals are in competition with each other, as Trm1 becomes mitochondrial if prevented from entering the nucleus.     

Nuclear envelope dynamics during mammalian spermatogenesis: new insights on male fertility

The production of highly specialized spermatozoa from undifferentiated spermatogonia is a strictly organized and programmed process requiring extensive restructuring of the entire cell. One of the most remarkable cellular transformations accompanying the various phases of spermatogenesis is the profound remodelling of the nuclear architecture, in which the nuclear envelope (NE) seems to be crucially involved. In recent years, several proteins from the distinct layers forming the NE (i.e. the inner and outer nuclear membranes as well as the nuclear lamina) have been associated with meiosis and/or spermiogenesis in different mammalian species. Among these are A‐ and B‐type lamins, Dpy‐19‐like protein 2 (DPY19L2), lamin B receptor (LBR), lamina‐associated polypeptide 1 (LAP1), LAP2/emerin/MAN1 (LEM) domain‐containing proteins, spermatogenesis‐associated 46 (SPATA46) and diverse elements of the linker of nucleoskeleton and cytoskeleton (LINC) complex, namely Sad‐1/UNC‐84 homology (SUN) and Klarsicht/ANC‐1/Syne‐1 homology (KASH) domain‐containing proteins. Herein, we summarize the current state of the art on the cellular and subcellular distribution of NE proteins expressed during mammalian spermatogenesis, and discuss the latest research developments regarding their testis‐specific functions. This review provides a comprehensive and innovative overview of the NE network as a regulatory platform and as an essential determinant of efficient meiotic chromosome recombination as well as spermiogenesis‐associated nuclear remodelling and differentiation in mammalian male germline cells. Thus, this review provides important novel insights on the biological relevance of NE proteins for male fertility.     

昆虫中的核输出现象

核质转运是真核细胞生命活动重要的组成部分,细胞核内蛋白的平衡及转录出的RNA出核成熟过程都依赖于核输出,核输出与真核生物的生命活动密切相关;病原物在侵染昆虫时也会利用宿主的核输出机制干扰宿主的免疫反应或劫持宿主的核输出蛋白以利于病毒的组装及复制.本文主要介绍真核细胞中核输出的基本机制、蚊子和果蝇等模式昆虫中核输出通路;...     

Review: The Dynamics of the Nuclear Lamins during the Cell Cycle— Relationship between Structure and Function

The nuclear lamins are members of the intermediate filament (IF) family of proteins. The lamins have an essential role in maintaining nuclear integrity, as do the other IF family members in the cytoplasm. Also like cytoplasmic IFs, the organization of lamins is dynamic. The lamins are found not only at the nuclear periphery but also in the interior of the nucleus, as distinct nucleoplasmic foci and possibly as a network throughout the nucleus. Nuclear processes such as DNA replication may be organized around these structures. In this review, we discuss changes in the structure and organization of the nuclear lamins during the cell cycle and during cell differentiation. These changes are correlated with changes in nuclear structure and function. For example, the interactions of lamins with chromatin and nuclear envelope components occur very early during nuclear assembly following mitosis. During S-phase, the lamins colocalize with markers of DNA replication, and proper lamin organization must be maintained for replication to proceed. When cells differentiate, the expression pattern of lamin isotypes changes. In addition, changes in lamin organization and expression patterns accompany the nuclear alterations observed in transformed cells. These lamin structures may modulate nuclear function in each of these processes.     

Binding Site Distribution of Nuclear Transport Receptors and Transport Complexes in Single Nuclear Pore Complexes

Transport through the nuclear pore complex (NPC) involves a large channel and an abundance of binding sites for nuclear transport receptors (NTRs). However, the mechanistically important distribution of NTR-binding sites along the channel is vividly debated. In this study, we visualized binding site distributions directly by two complementary optical super-resolution methods, single-molecule microscopy and 4Pi microscopy. First, we analyzed the distribution of RanGDP because this important nuclear transport substrate has two types of binding sites at the NPC, direct and indirect, NTR-mediated sites. We found that the direct binding sites had a maximum at approximately −30 nm with regard to the NPC center, whereas the indirect transport-relevant binding sites peaked at approximately −10 nm. The 20 nm-shift could be only resolved by 4Pi microscopy because of a two to threefold improved localization precision as compared with single-molecule microscopy. Then we analyzed the distribution of the NTR Kapβ1 and a Kapβ1-based transport complex and found them to have also binding maxima at approximately −10 nm. These observations support transport models in which NTR binding sites are distributed all along the transport channel and argue against models in which the cytoplasmic entrance of the channel is surrounded by a large cloud of binding sites.     

Nucleoporin FG Domains Facilitate mRNP Remodeling at the Cytoplasmic Face of the Nuclear Pore Complex

Directional export of messenger RNA (mRNA) protein particles (mRNPs) through nuclear pore complexes (NPCs) requires multiple factors. In Saccharomyces cerevisiae, the NPC proteins Nup159 and Nup42 are asymmetrically localized to the cytoplasmic face and have distinct functional domains: a phenylalanine-glycine (FG) repeat domain that docks mRNP transport receptors and domains that bind the DEAD-box ATPase Dbp5 and its activating cofactor Gle1, respectively. We speculated that the Nup42 and Nup159 FG domains play a role in positioning mRNPs for the terminal mRNP-remodeling steps carried out by Dbp5. Here we find that deletion (Δ) of both the Nup42 and Nup159 FG domains results in a cold-sensitive poly(A)+ mRNA export defect. The nup42ΔFG nup159ΔFG mutant also has synthetic lethal genetic interactions with dbp5 and gle1 mutants. RNA cross-linking experiments further indicate that the nup42ΔFG nup159ΔFG mutant has a reduced capacity for mRNP remodeling during export. To further analyze the role of these FG domains, we replaced the Nup159 or Nup42 FG domains with FG domains from other Nups. These FG “swaps” demonstrate that only certain FG domains are functional at the NPC cytoplasmic face. Strikingly, fusing the Nup42 FG domain to the carboxy-terminus of Gle1 bypasses the need for the endogenous Nup42 FG domain, highlighting the importance of proximal positioning for these factors. We conclude that the Nup42 and Nup159 FG domains target the mRNP to Gle1 and Dbp5 for mRNP remodeling at the NPC. Moreover, these results provide key evidence that character and context play a direct role in FG domain function and mRNA export.     

Breaking and making of the nuclear envelope

During mitosis, a single nucleus gives rise to two nuclei that are identical to the parent nucleus. Mitosis consists of a continuous sequence of events that must be carried out once and only once. Two such important events are the disassembly of the nuclear envelope (NE) during the first stages of mitosis, and its accurate reassembly during the last stages of mitosis. NE breakdown (NEBD) is initiated when maturation-promoting factor (MPF) enters the nucleus and starts phosphorylating nuclear pore complexes (NPCs) and nuclear lamina proteins, followed by NPC and lamina breakdown. Nuclear reassembly starts when nuclear membranes assemble onto the chromatin. This article focuses on the different models of NEBD and reassembly with emphasis on recent data.     

Age- and sex-related differences in nuclear lipid content and nucleoside triphosphatase activity in the JCR:LA-cp corpulent rat

The putative role of the nuclear nucleoside triphosphatase (NTPase) is to provide energy to the nuclear pore complex for poly A(+) mRNA export. Previous work has demonstrated that liver nuclear NTPase activity is greater in 6 month old corpulent (cp/cp) female JCR:LA rats, a hyperlipidemic rat model, compared to lean (+/?) animals. This increase appeared to be related to increases in nuclear membrane cholesterol content. The current study extended these initial data to compare NTPase activity as a function of age and sex in isolated JCR:LA-cp rat liver nuclei, to further test the hypothesis that nuclear membrane cholesterol may modulate NTPase activity. NTPase activity was increased in cp/cp female animals compared to +/? females at all ages studied, with V_max values increased by 60-176%. Membrane integrity of cp/cp female nuclei was reduced compared to +/? female nuclei. Nuclear membrane cholesterol levels increased linearly with age by 50, 150 and 250% in 3, 6 and 9 month old cp/cp females over leans. In contrast, nuclei from cp/cp males exhibited only minor, isolated changes in NTPase activity. Furthermore, there were no significant changes in nuclear cholesterol content or membrane integrity in the less hyperlipidemic male animals at any age. These data suggest that altered lipid metabolism may lead to changes in nuclear membrane structure, which in turn may alter NTPase activity and functioning of the nuclear pore complex.

     

Core Components of the Nuclear Pore Bind Distinct States of Chromatin and Contribute to Polycomb Repression

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Early localization of NPA58, a rat nuclear pore-associated protein, to the reforming nuclear envelope during mitosis

We have studied the mitotic reassembly of the nuclear envelope, using antibodies to nuclear marker proteins and NPA58 in F-111 rat fibroblast cells. In earlier studies we have proposed that NPA58, a 58 kDa rat nuclear protein, is involved in nuclear protein import. In this report, NPA58 is shown to be localized on the cytoplasmic face of the envelope in interphase cells, in close association with nuclear pores. In mitotic cells NPA58 is dispersed in the cytoplasm till anaphase. The targeting of NPA58 to the reforming nuclear envelope in early telophase coincides with the recruitment of a well-characterized class of nuclear pore proteins recognized by the antibody mAb 414, and occurs prior to the incorporation of lamin B1 into the envelope. Significant protein import activity is detectable only after localization of NPA58 in the newly-formed envelope. The early targeting of NPA58 is consistent with its proposed role in nuclear transport.     

A classical NLS and the SUN domain contribute to the targeting of SUN2 to the inner nuclear membrane

Integral membrane proteins of the inner nuclear membrane (INM) are inserted into the endoplasmic reticulum membrane during their biogenesis and are then targeted to their final destination. We have used human SUN2 to delineate features that are required for INM targeting and have identified multiple elements that collectively contribute to the efficient localization of SUN2 to the nuclear envelope (NE). One such targeting element is a classical nuclear localization signal (cNLS) present in the N‐terminal, nucleoplasmic domain of SUN2. A second motif proximal to the cNLS is a cluster of arginines that serves coatomer‐mediated retrieval of SUN2 from the Golgi. Unexpectedly, also the C‐terminal, lumenal SUN domain of SUN2 supports NE localization, showing that targeting elements are not limited to cytoplasmic or transmembrane domains of INM proteins. Together, SUN2 represents the first mammalian INM protein relying on a functional cNLS, a Golgi retrieval signal and a perinuclear domain to mediate targeting to the INM.     

Nuclear import of the ran exchange factor, RCC1, is mediated by at least two distinct mechanisms

RCC1, the only known guanine-nucleotide exchange factor for the Ran GTPase, is an approximately 45-kD nuclear protein that can bind chromatin. An important question concerns how RCC1 traverses the nuclear envelope. We now show that nuclear RCC1 is not exported readily in interphase cells and that the import of RCC1 into the nucleoplasm is extremely rapid. Import can proceed by at least two distinct mechanisms. The first is a classic import pathway mediated by basic residues within the NH(2)-terminal domain (NTD) of RCC1. This pathway is dependent upon both a preexisting Ran gradient and energy, and preferentially uses the importin-alpha3 isoform of importin-alpha. The second pathway is not mediated by the NTD of RCC1. This novel pathway does not require importin-alpha or importin-beta or the addition of any other soluble factor in vitro; however, this pathway is saturable and sensitive only to a subset of inhibitors of classical import pathways. Furthermore, the nuclear import of RCC1 does not require a preexisting Ran gradient or energy. We speculate that this second import pathway evolved to ensure that RCC1 never accumulates in the cytoplasm.     

Nuclear membrane cholesterol can modulate nuclear nucleoside triphosphatase activity

Previous work has suggested that changes in nuclear membrane cholesterol may induce a stimulation in nuclear nucleoside triphosphatase (NTPase) activity. The purpose of the present study was to directly investigate if nuclear membrane cholesterol can stimulate nuclear NTPase activity. The cholesterol content of nuclei was altered with a liposomal methodology. The cholesterol content of nuclei isolated from hepatic tissue was relatively low in comparison to that typically exhibited by other membrane fractions. Because of this, it was difficult to further deplete the nuclear membrane of cholesterol, but we could successfully increase the cholesterol content after exposure to cholesterol-enriched liposomes. Nuclear NTPase activity was potently stimulated (∼︁ 150–200% of control) by an increase in the nuclear membrane cholesterol content. The Vmax of the NTPase activity in the presence of ATP or GTP was significantly increased after cholesterol enrichment without altering the affinity of the enzyme for these moieties. Mg²⁺ dependency of NTPase activity was also altered by cholesterol incorporation into the nuclear membrane. Cholesterol enrichment of the nuclear membrane also left the nuclei more susceptible to damage by salt-induced lysis than control nuclei. Our results clearly demonstrate that the cholesterol content of the nuclear membrane will have significant, direct effects on nuclear integrity and NTPase activity. © 1996 Wiley-Liss, Inc.     

Nuclear transport and nuclear pores in yeast

The central features of nuclear import have been conserved during evolution. In yeast the nuclear accumulation of proteins follows the same selective and active transport mechanisms known from higher eukaryotes. Yeast nuclear proteins contain nuclear localization sequences (NLS) which are presumably recognized by receptors in the cytoplasm and the nuclear envelope. Subsequent to this recognition step, nuclear proteins are translocated into the nucleus via the nuclear pore complexes. The structure of the yeast nuclear pore complex resembles that of higher eukaryotes. Recently, the first putative components of the yeast nuclear import machinery have been cloned and sequenced. The genetically amenable yeast system allows for an efficient structural and functional analysis of these components. Due to the evolutionary conservation potential insights into the nuclear import mechanisms in yeast can be transferred to higher eukaryotes. Thus, yeast can be considered as a eukaryotic model system to study nuclear transport.