The nuclear pore complex (NPC) and its relationship to the nuclear envelope (NE) was characterized in living cells using POM121–green fluorescent protein (GFP) and GFP-Nup153, and GFP–lamin B1. No independent movement of single pore complexes was found within the plane of the NE in interphase. Only large arrays of NPCs moved slowly and synchronously during global changes in nuclear shape, strongly suggesting mechanical connections which form an NPC network. The nuclear lamina exhibited identical movements. NPC turnover measured by fluorescence recovery after photobleaching of POM121 was less than once per cell cycle. Nup153 association with NPCs was dynamic and turnover of this nucleoporin was three orders of magnitude faster. Overexpression of both nucleoporins induced the formation of annulate lamellae (AL) in the endoplasmic reticulum (ER). Turnover of AL pore complexes was much higher than in the NE (once every 2.5 min). During mitosis, POM121 and Nup153 were completely dispersed and mobile in the ER (POM121) or cytosol (Nup153) in metaphase, and rapidly redistributed to an immobilized pool around chromatin in late anaphase. Assembly and immobilization of both nucleoporins occurred before detectable recruitment of lamin B1, which is thus unlikely to mediate initiation of NPC assembly at the end of mitosis.

Abstract.We have identified an integral membrane protein of 145 kD (estimated by SDS-PAGE) of rat liver nuclear envelopes that binds to WGA. We obtained peptide sequence from purified p145 and cloned and sequenced several eDNA clones and one genomic clone. The relative molecular mass of p145 calculated from its complete, eDNA deduced primary structure is 120.7 kD. Antibodies raised against a synthetic peptide represented in p145 reacted monospecifically with p145. In indirect immunofluorescence these antibodies gave punctate staining of the nuclear envelope. Immunogold EM showed specific decoration of the nuclear pores. Thus p145 is an integral membrane protein located specifically in the "pore membrane" domain of the nuclear envelope. To indicate this specific location, and based on its calculated relative molecular mass, the protein is termed POM i21 (pore membrane protein of 12I kD). The 1,199-residue-long primary structure shows a hydrophobic region (residues 29-72) that is likely to form one (or two adjacent) transmembrane segment(s). The bulk of the protein (residues 73-1199) is predicted to be exposed not on the cisternal side but on the pore side of the pore membrane. It contains 36 consensus sites for various kinases. However, its most striking feature is a repetitive pentapeptide motif XFXFG that has also been shown to occur in several nucleoporins. This nucleoporin-like domain of POM 121 is proposed to function in anchoring components of the nuclear pore complex to the pore membrane.T HE nuclear envelope (NE) ~ consists of three morphologically and biochemically distinct domains. The outer nuclear membrane with its attached ribosomes is continuous with the RER. One of the principal functions of this membrane system is to serve as the port of entry for all proteins, soluble and membrane integrated, destined for the membranes and compartments of the exocytotic and endocytotic pathway. The inner nuclear membrane is attached to the nuclear lamina and/or chromatin components and has been proposed to serve in the three-dimensional organization of chromatin (1). At numerous circumscribed points, the outer and inner nuclear membranes are connected with each other forming circular nuclear pores of •100 nm diameter. These connecting bits of membrane appear to be biochemicaUy and functionally distinct from both the outer and inner nuclear membrane and therefore can be regarded as a distinct third domain of the nuclear envelope referred to as the "pore membrane: The large nuclear pore complexes (NPCs) (estimated mass of 1.25 x 108 Daltons) (27) occupy the nuclear pores. There are as many pore membrane domains in a single nuclear envelope as there are NPCs.E. Hallberg's present address is Department of Biochemistry, Arrheniuslaboratory, Stockholm University, S-106 91, Stockholm, Sweden.

The HIV-1 genome contains several genes coding for auxiliary proteins, including the small Vpr protein. Vpr affects the integrity of the nuclear envelope and participates in the nuclear translocation of the preintegration complex containing the viral DNA. Here, we show by photobleaching experiments performed on living cells expressing a Vpr-green fluorescent protein fusion that the protein shuttles between the nucleus and the cytoplasm, but a significant fraction is concentrated at the nuclear envelope, supporting the hypothesis that Vpr interacts with components of the nuclear pore complex. An interaction between HIV-1 Vpr and the human nucleoporin CG1 (hCG1) was revealed in the yeast twohybrid system, and then confirmed both in vitro and in transfected cells. This interaction does not involve the FG repeat domain of hCG1 but rather the N-terminal region of the protein. Using a nuclear import assay based on digitonin-permeabilized cells, we demonstrate that hCG1 participates in the docking of Vpr at the nuclear envelope. This association of Vpr with a component of the nuclear pore complex may contribute to the disruption of the nuclear envelope and to the nuclear import of the viral DNA.In contrast to oncoretroviruses that replicate only in dividing cells and enter the nucleus upon nuclear breakdown during mitosis, HIV-1 1 and other lentiviruses have the ability to infect non-dividing cells, such as macrophages and quiescent T lymphocytes. After entry of the virus into the cell, the HIV capsid seems to uncoat rapidly. The genomic HIV-1 RNA is reverse transcribed into linear double-stranded DNA, which remains associated with a nucleoprotein complex, called the preintegration complex (PIC). The viral DNA is then imported into the nucleus through the nuclear envelope (NE) via an active mechanism within 4 -6 h after infection (1).In eukaryotic cells, the NE creates distinct nuclear and cytoplasmic compartments. This structure consists of two concentric membranes, the inner and outer nuclear membranes which are continuous with the endoplasmic reticulum. The NE is stabilized by the nuclear lamina, a tight meshwork of intermediate filament proteins underlying the inner nuclear membrane (for review, see Ref.2), whereas on the outer side, cytoplasmic intermediate filaments in close contact with the nucleus serve to suspend the nucleus in the cytoplasm (3). Spanning both membranes are nuclear pore complexes (NPC) that form aqueous channels, which allow selective traffic between nucleus and cytoplasm and impose a permeability barrier to free diffusion of macromolecules or complexes. The NPC is a large supramolecular structure (4) formed of ϳ30 unique proteins in vertebrates, termed nucleoporins (Nups) giving rise to an estimated molecular mass of 60 MDa (Ref. 5; for reviews, see Refs. 6 and 7). High resolution electron microscopic images of NPCs reveal an 8-fold symmetric structure, formed by nuclear and cytoplasmic rings and a central spoke complex. Peripheral filaments emanate from the core of the complex into the nucle...

Here, we characterize a transmembrane protein of the nuclear envelope that we name spindle-associated membrane protein 1 (Samp1). The protein is conserved in metazoa and fission yeast and is homologous to Net5 in rat and Ima1 in Schizosaccharomyces pombe. We show that, in human cells, the protein is a membrane-spanning polypeptide with an apparent molecular mass of 43 kDa. This is consistent with a predicted polypeptide of 392 amino acids that has five transmembrane segments and its C-terminus exposed to the nucleoplasm. During interphase, Samp1 was specifically distributed in the inner nuclear membrane. Post-transcriptional silencing of Samp1 expression resulted in separation of centrosomes from the nuclear envelope, indicating that it is functionally connected to the cytoskeleton. At the onset of mitosis, most of the protein dispersed out into the ER, as expected. However, during mitosis, a significant fraction of the protein specifically localized to the polar regions of the mitotic spindle. We demonstrate for the first time, in human cells, the existence of a membranous structure overlapping with the mitotic spindle. Interestingly, another integral inner nuclear membrane protein, emerin, was absent from the spindle-associated membranes. Thus, Samp1 defines a specific membrane domain associated with the mitotic spindle.