Günter Blobel

The nuclear envelope lamina is reversibly depolymerized during mitosis

The nuclear envelope lamina is a supramolecular protein assembly associated with the nucleoplasmic surface of the inner nuclear membrane, which contains three predominant polypeptide components in mammalian cells (lamins A, B and C). We previously demonstrated by immunofluorescence microscopy that the lamina is reversibly disassembled during cell division, coincident with the disassembly and reconstruction of the mitotic nuclear envelope architecture. In this paper, these immunocytochemical observations are extended with cell fractionation and immunoprecipitation studies performed on synchronized populations of tissue culture cells. With these techniques, we have established that during mitosis, lamina A and C occur in a soluble and nonmembrane-associated state. In contrast, the mitotic lamin B may be associated with membrane fragments derived from the disassembled interphase nuclear envelope. From sedimentation analysis on sucrose gradients, we have determined that all three lamins are monomeric at periods of mitotic lamina disassembly. These results, together with quantitative immunoprecipitation studies, demonstrate that the lamina is reversibly depolymerized during cell division. Attendant with the depolymerized state of the lamina, the mitotic lamins (which are phosphoproteins) have a distinctly more acidic isoelectric point and a substantially higher level of phosphorylation compared to their interphase counterparts. This indicates that reversible enzymatic phosphorylations of the lamins may be involved in modulating the state of polymerization of the lamina and its reversible mitotic disassembly.

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.

Protein translocation across the endoplasmic reticulum

In the past year, dramatic progress has been made in our understanding of protein biogenesis at the initial steps of the eukaryotic secretory pathway. New insights have refined our view of protein targeting to the endoplasmic reticulum membrane and provided the best glimpse so far of the subsequent translocation step. The interactions of three GTP-binding proteins have been found to result in a novel cycle of GTP binding and hydrolysis to regulate protein targeting. Experiments with fluorescent probes have revealed that the nascent chain enters an aqueous environment within the membrane sealed off from the cytosol. In vitro reconstitution experiments have shown surprising simplicity in the number of polypeptides required to facilitate translocation across a synthetic membrane and to promote the integration of membrane proteins. Furthermore, new genetic and functional similarities between divergent organisms have been discovered, providing convincing evidence of the evolutionary conservation of strategies used in the targeting and translocation of polypeptides.

Identification and characterization of a nuclear pore complex protein

We describe studies using a monoclonal antibody that recognizes a 62 kd protein (p62) of rat liver nuclei. This protein remains associated with the nuclear pore complex-lamina fraction resulting from treatment of nuclei with DNAase, RNAase, and nonionic detergent. Immunofluorescence revealed a strikingly punctate pattern of nuclear rim staining. By immunoferritin microscopy, p62 was specifically localized to the pore complex. Thus, pore complexes can be resolved by fluorescence light microscopy. Pulse chase analysis of labeled tissue culture cells showed that p62 is synthesized as a soluble cytoplasmic precursor of 61 kd, which is incorporated into the nuclear fraction with an unusually long t1/2 of about 6 hr. Incorporation is followed by modification that may involve addition of N-acetylglucosamine residues.

[6] Preparation of microsomal membranes for cotranslational protein translocation

Publisher Summary This chapter describes a rapid isolation procedure that reproducibly yields highly active microsomal membranes, and presents method for refining the crude rough microsomes (RM) fraction by column washing, EDTA stripping, or nuclease treatment. The column-washed RM has almost no inhibitory effects on protein synthesis; EDTA-stripped RM has a high specific activity, because of removal of ribosomes and proteins; nuclease-treated RM has essentially no endogenous mRNA activity. All these microsome preparations still contain signal recognition particle (SRP). They are, therefore, translocation-active in wheat germ as well as reticulocyte lysate. Most of the microsome preparations are glycosylation active, but there is a high variability from preparation to preparation in the extent of glycosylation obtained. Microsomal membranes from sources other than dog pancreas are also employed in cotranslational studies, and the most successfully used alternative systems are probably chicken oviduct RM and adrenal microsomes.

The ubiquitin‐like protein Smt3p is activated for conjugation to other proteins by an Aos1p/Uba2p heterodimer

SMT3 is an essential Saccharomyces cerevisiae gene encoding a 11.5 kDa protein similar to the mammalian ubiquitin‐like protein SUMO‐1. We have found that Smt3p, like SUMO‐1 and ubiquitin, can be attached to other proteins post‐translationally and have characterized the processes leading to the activation of the Smt3p C‐terminus for conjugation. First, the SMT3 translation product is cleaved endoproteolytically to expose Gly98, the mature C‐terminus. The presence of Gly98 is critical for Smt3ps abilities to be conjugated to protein substrates and to complement the lethality of a smt3Δ strain. Smt3p undergoes ATP‐dependent activation by a novel heterodimeric enzyme consisting of Uba2p, a previously identified 71 kDa protein similar to the C‐terminus of ubiquitin‐activating enzymes (E1s), and Aos1p (activation of Smt3p), a 40 kDa protein similar to the N‐terminus of E1s. Experiments with conditional uba2 mutants showed that Uba2p is required for Smt3p conjugation in vivo. Furthermore, UBA2 and AOS1 are both essential genes, providing additional evidence that they act in a distinct pathway whose role in cell viability is to conjugate Smt3p to other proteins.

Structure of the 80S Ribosome from Saccharomyces cerevisiae—tRNA-Ribosome and Subunit-Subunit Interactions

A cryo-EM reconstruction of the translating yeast 80S ribosome was analyzed. Computationally separated rRNA and protein densities were used for docking of appropriately modified rRNA models and homology models of yeast ribosomal proteins. The core of the ribosome shows a remarkable degree of conservation. However, some significant differences in functionally important regions and dramatic changes in the periphery due to expansion segments and additional ribosomal proteins are evident. As in the bacterial ribosome, bridges between the subunits are mainly formed by RNA contacts. Four new bridges are present at the periphery. The position of the P site tRNA coincides precisely with its prokaryotic counterpart, with mainly rRNA contributing to its molecular environment. This analysis presents an exhaustive inventory of an eukaryotic ribosome at the molecular level.

Ubc9p Is the Conjugating Enzyme for the Ubiquitin-like Protein Smt3p

At least one essential function of Smt3p, aSaccharomyces cerevisiae ubiquitin-like protein similar to the mammalian protein SUMO-1, involves its posttranslational covalent attachment to other proteins. Using Smt3p affinity chromatography, we have isolated the second enzyme of the Smt3p conjugation pathway and have found that it is identical to Ubc9p, a previously identified protein that has extensive sequence similarity to the ubiquitin-conjugating enzymes (E2s) and that is required for yeast to progress through mitosis. A hallmark of E2s is the ability to form a thioester bond-containing covalent intermediate with ubiquitin (Ub). While we were unable to detect formation of a Ub∼Ubc9p thioester, Ubc9p was found to form a thioester with Smt3p, indicating that Ubc9p is the functional analog of E2s in the Smt3p pathway and that this step is distinct from the ubiquitin pathway. Ubc9p is required for attachment of Smt3p to other proteins in vitro, suggesting that it is the only such enzyme in S. cerevisiae. These results suggest that, like ubiquitination, Smt3p conjugation may be a critical modification in cell cycle regulation.

Karyopherins and nuclear import.

Proteins of the karyopherin alpha and karyopherin beta families play a central role in nucleocytoplasmic transport. Recently, crystal structures of karyopherin alpha and its complexes with nuclear localization signal peptides, a karyopherin beta2-Ran complex and complexes of full-length and fragments of karyopherin beta1 with import substrates, Ran and nucleoporins have been solved. These karyopherin structures provide valuable insights into understanding the molecular mechanism of nuclear import, especially substrate recognition, substrate release by GTPase and interactions with the nuclear pore complex.

The Peptide Repeat Domain of Nucleoporin Nup98 Functions as a Docking Site in Transport across the Nuclear Pore Complex

We report the cDNA deduced primary structure of a wheat germ agglutinin-reactive nuclear pore complex (NPC) protein of rat. The protein, termed Nup98 (for nucleoporin of 98 kDa), contains numerous GLFG and FG repeats and some FXFG repeats and is thus a vertebrate member of a family of GLFG nucleoporins that were previously discovered in yeast. Immunoelectron microscopy showed Nup98 to be asymmetrically located at the nucleoplasmic side of the NPC. Nup98 functions as one of several docking site nucleoporins in a cytosolic docking activity-mediated binding of a model transport substrate. The docking site of Nup98 was mapped to its N-terminal half, which contains all of the peptide repeats. A recombinant segment of this region depleted the docking activity of cytosol. We suggest that the peptide repeat domain of Nup98, together with peptide repeat domains of other nucleoporins, forms an array of sites for mediated docking of transport substrate, and that bidirectional transport across the NPC proceeds by repeated docking and undocking reactions.