Michael Rexach

COPII: a membrane coat formed by Sec proteins that drive vesicle budding from the endoplasmic reticulum.

In vitro synthesis of endoplasmic reticulum-derived transport vesicles has been reconstituted with washed membranes and three soluble proteins (Sar1p, Sec13p complex, and Sec23p complex). Vesicle formation requires GTP but can be driven by nonhydrolyzable analogs such as GMP-PNP. However, GMP-PNP vesicles fail to target and fuse with the Golgi complex whereas GTP vesicles are functional. All the cytosolic proteins required for vesicle formation are retained on GMP-PNP vesicles, while Sar1p dissociates from GTP vesicles. Thin section electron microscopy of purified preparations reveals a uniform population of 60-65 nm vesicles with a 10 nm thick electron dense coat. The subunits of this novel coat complex are molecularly distinct from the constituents of the nonclathrin coatomer involved in intra-Golgi transport. Because the overall cycle of budding driven by these two types of coats appears mechanistically similar, we propose that the coat structures be called COPI and COPII.

Protein import into nuclei: association and dissociation reactions involving transport substrate, transport factors, and nucleoporins

The molecular dynamics of nuclear protein import were examined in a solution binding assay by testing for interactions between a protein containing a nuclear localization signal (NLS), the transport factors karyopherin alpha, karyopherin beta, and Ran, and FXFG or GLFG repeat regions of nucleoporins. We found that karyopherins alpha and beta cooperate to bind FXFG but not GLFG repeat regions. Binding of the NLS protein to karyopherin alpha was enhanced by karyopherin beta. Two novel reactions were discovered. First, incubation of a karyopherin heterodimer-NLS protein complex with an FXFG repeat region stimulated the dissociation of the NLS protein from the karyopherin heterodimer. Second, incubation of the karyopherin heterodimer with RanGTP (or with a Ran mutant that cannot hydrolyze GTP) led to the dissociation of karyopherin alpha from beta and to an association of Ran with karyopherin beta; RanGDP had no effect. We propose that movement of NLS proteins across the nuclear pore complex is a stochastic process that operates via repeated association-dissociation reactions.

Disorder in the nuclear pore complex: The FG repeat regions of nucleoporins are natively unfolded

Nuclear transport proceeds through nuclear pore complexes (NPCs) that are embedded in the nuclear envelope of eukaryotic cells. The Saccharomyces cerevisiae NPC is comprised of 30 nucleoporins (Nups), 13 of which contain phenylalanine-glycine repeats (FG Nups) that bind karyopherins and facilitate the transport of karyopherin-cargo complexes. Here, we characterize the structural properties of S. cerevisiae FG Nups by using biophysical methods and predictive amino acid sequence analyses. We find that FG Nups, particularly the large FG repeat regions, exhibit structural characteristics typical of “natively unfolded” proteins (highly flexible proteins that lack ordered secondary structure). Furthermore, we use protease sensitivity assays to demonstrate that most FG Nups are disordered in situ within the NPCs of purified yeast nuclei. The conclusion that FG Nups constitute a family of natively unfolded proteins supports the hypothesis that the FG repeat regions of Nups form a meshwork of random coils at the NPC through which nuclear transport proceeds.

Natively Unfolded Nucleoporins Gate Protein Diffusion across the Nuclear Pore Complex

Nuclear pore complexes (NPCs) form aqueous conduits in the nuclear envelope and gate the diffusion of large proteins between the cytoplasm and nucleoplasm. NPC proteins (nucleoporins) that contain phenylalanine-glycine motifs in filamentous, natively unfolded domains (FG domains) line the diffusion conduit of the NPC, but their role in the size-selective barrier is unclear. We show that deletion of individual FG domains in yeast relaxes the NPC permeability barrier. At the molecular level, the FG domains of five nucleoporins anchored at the NPC center form a cohesive meshwork of filaments through hydrophobic interactions, which involve phenylalanines in FG motifs and are dispersed by aliphatic alcohols. In contrast, the FG domains of four peripherally anchored nucleoporins are generally noncohesive. The results support a two-gate model of NPC architecture featuring a central diffusion gate formed by a meshwork of cohesive FG nucleoporin filaments and a peripheral gate formed by repulsive FG nucleoporin filaments.

Viral protein R regulates nuclear import of the HIV‐1 pre‐integration complex

Replication of human immunodeficiency virus type 1 (HIV‐1) in non‐dividing cells critically depends on import of the viral pre‐integration complex into the nucleus. Genetic evidence suggests that viral protein R (Vpr) and matrix antigen (MA) are directly involved in the import process. An in vitro assay that reconstitutes nuclear import of HIV‐1 pre‐integration complexes in digitonin‐permeabilized cells was used to demonstrate that Vpr is the key regulator of the viral nuclear import process. Mutant HIV‐1 pre‐integration complexes that lack Vpr failed to be imported in vitro, whereas mutants that lack a functional MA nuclear localization sequence (NLS) were only partially defective. Strikingly, the import defect of the Vpr− mutant was rescued when recombinant Vpr was re‐added. In addition, import of Vpr− virus was rescued by adding the cytosol of HeLa cells, where HIV‐1 replication had been shown to be Vpr‐independent. In a solution binding assay, Vpr associated with karyopherin α, a cellular receptor for NLSs. This association increased the affinity of karyopherin α for basic‐type NLSs, including that of MA, thus explaining the positive effect of Vpr on nuclear import of the HIV‐1 pre‐integration complex and BSA–NLS conjugates. These results identify the biochemical mechanism of Vpr function in transport of the viral pre‐integration complex to, and across, the nuclear membrane.

Reconstitution of SEC gene product-dependent intercompartmental protein transport.

Transport of alpha-factor precursor from the endoplasmic reticulum to the Golgi apparatus has been reconstituted in gently lysed yeast spheroplasts. Transport is measured through the coupled addition of outer-chain carbohydrate to [35S]methionine-labeled alpha-factor precursor translocated into the endoplasmic reticulum of broken spheroplasts. The reaction is absolutely dependent on ATP, stimulated 6-fold by cytosol, and occurs between physically separable sealed compartments. Transport is inhibited by the guanine nucleotide analog GTP gamma S. sec23 mutant cells have a temperature-sensitive defect in endoplasmic reticulum-to-Golgi transport in vivo. This defect has been reproduced in vitro using sec23 membranes and cytosol. Transport at 30 degrees C with sec23 membranes requires addition of cytosol containing the SEC23 (wild-type) gene product. This demonstrates that an in vitro inter-organelle transport reaction depends on a factor required for transport in vivo. Complementation of sec mutations in vitro provides a functional assay for the purification of individual intercompartmental transport factors.

A bimodal distribution of two distinct categories of intrinsically-disordered structures with separate functions in FG nucleoporins

Nuclear pore complexes (NPCs) gate the only conduits for nucleocytoplasmic transport in eukaryotes. Their gate is formed by nucleoporins containing large intrinsically disordered domains with multiple phenylalanine-glycine repeats (FG domains). In combination, these are hypothesized to form a structurally and chemically homogeneous network of random coils at the NPC center, which sorts macromolecules by size and hydrophobicity. Instead, we found that FG domains are structurally and chemically heterogeneous. They adopt distinct categories of intrinsically disordered structures in non-random distributions. Some adopt globular, collapsed coil configurations and are characterized by a low charge content. Others are highly charged and adopt more dynamic, extended coil conformations. Interestingly, several FG nucleoporins feature both types of structures in a bimodal distribution along their polypeptide chain. This distribution functionally correlates with the attractive or repulsive character of their interactions with collapsed coil FG domains displaying cohesion toward one another and extended coil FG domains displaying repulsion. Topologically, these bipartite FG domains may resemble sticky molten globules connected to the tip of relaxed or extended coils. Within the NPC, the crowding of FG nucleoporins and the segregation of their disordered structures based on their topology, dimensions, and cohesive character could force the FG domains to form a tubular gate structure or transporter at the NPC center featuring two separate zones of traffic with distinct physicochemical properties.

Disassembly of RanGTP-karyopherin beta complex, an intermediate in nuclear protein import

We previously showed that RanGTP forms a 1:1 complex with karyopherin β that renders RanGTP inaccessible to RanGAP (Floer, M., and Blobel, G. (1996) J. Biol. Chem. 271, 5313–5316) and karyopherin β functionally inactive (Rexach, M., and Blobel, G. (1995) Cell 83, 683–692). Recycling of both factors for another round of function requires dissociation of the RanGTP-karyopherin β complex. Here we show using BIAcore™, a solution binding assay, and GTP hydrolysis and exchange assays, with yeast proteins, that karyopherin β and RanGTP are recycled efficiently in a reaction that involves karyopherin α, RanBP1, RanGAP, and the C terminus of the nucleoporin Nup1. We find that karyopherin α first releases RanGTP from karyopherin β in a reaction that does not require GTP hydrolysis. The released RanGTP is then sequestered by RanBP1, and the newly formed karyopherin αβ binds to the C terminus of Nup1. Finally, RanGTP is converted to RanGDP via nucleotide hydrolysis when RanGAP is present. Conversion of RanGTP to RanGDP can also occur via nucleotide exchange in the presence of RanGEF, an excess of GDP, and if RanBP1 is absent. Additional nucleoporin domains that bind karyopherin αβ stimulate recycling of karyopherin β and Ran in a manner similar to the C terminus of Nup1.

Comprehensive Analysis of a Multidimensional Liquid Chromatography Mass Spectrometry Dataset Acquired on a Quadrupole Selecting, Quadrupole Collision Cell, Time-of-flight Mass Spectrometer II. New Developments in Protein Prospector Allow for Reliable and Comprehensive Automatic Analysis of Large Datasets

A thorough analysis of the protein interaction partners of the yeast GTPase Gsp1p was carried out by a multidimensional chromatography strategy of strong cation exchange fractionation of peptides followed by reverse phase LC-ESI-MSMS using a QSTAR instrument. This dataset was then analyzed using the latest developmental version of Protein Prospector. The Prospector search results were also compared with results from the search engine “Mascot” using a new results comparison program within Prospector named “SearchCompare.” The results from this study demonstrate that the high quality data produced on a quadrupole selecting, quadrupole collision cell, time-of-flight (QqTOF) geometry instrument allows for confident assignment of the vast majority of interpretable spectra by current search engines.

The Saccharomyces cerevisiae Nucleoporin Nup2p Is a Natively Unfolded Protein

Little is known about the structure of the individual nucleoporins that form eukaryotic nuclear pore complexes (NPCs). We report here in vitro physical and structural characterizations of a full-length nucleoporin, the Saccharomyces cerevisiae protein Nup2p. Analyses of the Nup2p structure by far-UV circular dichroism (CD) spectroscopy, Fourier transform infrared (FTIR) spectroscopy, protease sensitivity, gel filtration, and sedimentation velocity experiments indicate that Nup2p is a “natively unfolded protein,” belonging to a class of proteins that exhibit little secondary structure, high flexibility, and low compactness. Nup2p possesses a very large Stokes radius (79 Å) in gel filtration columns, sediments slowly in sucrose gradients as a 2.9 S particle, and is highly sensitive to proteolytic digestion by proteinase K; these characteristics suggest a structure of low compactness and high flexibility. Spectral analyses (CD and FTIR spectroscopy) provide additional evidence that Nup2p contains extensive regions of structural disorder with comparatively small contributions of ordered secondary structure. We address the possible significance of natively unfolded nucleoporins in the mechanics of nucleocytoplasmic trafficking across NPCs.