Direct membrane protein-DNA interactions required early in nuclear envelope assembly

Among the earliest events in postmitotic nuclear envelope (NE) assembly are the interactions between chromatin and the membranes that will fuse to form the NE. It has been proposed that interactions between integral NE proteins and chromatin proteins mediate initial membrane recruitment to chromatin. We show that several transmembrane NE proteins bind to DNA directly and that NE membrane proteins as a class are enriched in long, basic domains that potentially bind DNA. Membrane fractions that are essential for NE formation are shown to bind directly to protein-free DNA, and our data suggest that these interactions are critical for early steps in NE assembly.     

Nup53 is required for nuclear envelope and nuclear pore complex assembly

Transport across the nuclear envelope (NE) is mediated by nuclear pore complexes (NPCs). These structures are composed of various subcomplexes of proteins that are each present in multiple copies and together establish the eightfold symmetry of the NPC. One evolutionarily conserved subcomplex of the NPC contains the nucleoporins Nup53 and Nup155. Using truncation analysis, we have defined regions of Nup53 that bind to neighboring nucleoporins as well as those domains that target Nup53 to the NPC in vivo. Using this information, we investigated the role of Nup53 in NE and NPC assembly using Xenopus egg extracts. We show that both events require Nup53. Importantly, the analysis of Nup53 fragments revealed that the assembly activity of Nup53 depleted extracts could be reconstituted using a region of Nup53 that binds specifically to its interacting partner Nup155. On the basis of these results, we propose that the formation of a Nup53-Nup155 complex plays a critical role in the processes of NPC and NE assembly.     

Two distinct interacting classes of nuclear envelope-associated coiled-coil proteins are required for the tissue-specific nuclear envelope targeting of Arabidopsis RanGAP

Ran GTPase plays essential roles in multiple cellular processes, including nucleocytoplasmic transport, spindle formation, and postmitotic nuclear envelope (NE) reassembly. The cytoplasmic Ran GTPase activating protein RanGAP is critical to establish a functional RanGTP/RanGDP gradient across the NE and is associated with the outer surface of the NE in metazoan and higher plant cells. Arabidopsis thaliana RanGAP association with the root tip NE requires a family of likely plant-specific nucleoporins combining coiled-coil and transmembrane domains (CC-TMD) and WPP domain-interacting proteins (WIPs). We have now identified, by tandem affinity purification coupled with mass spectrometry, a second family of CC-TMD proteins, structurally similar, yet clearly distinct from the WIP family, that is required for RanGAP NE association in root tip cells. A combination of loss-of-function mutant analysis and protein interaction data indicates that at least one member of each NE-associated CC-TMD protein family is required for RanGAP targeting in root tip cells, while both families are dispensable in other plant tissues. This suggests an unanticipated complexity of RanGAP NE targeting in higher plant cells, contrasting both the single nucleoporin anchor in metazoans and the lack of targeting in fungi and proposes an early evolutionary divergence of the underlying plant and animal mechanisms.     

GTP hydrolysis is required for vesicle fusion during nuclear envelope assembly in vitro

Nuclear envelope assembly was studied in vitro using extracts from Xenopus eggs. Nuclear-specific vesicles bound to demembranated sperm chromatin but did not fuse in the absence of cytosol. Addition of cytosol stimulated vesicle fusion, pore complex assembly, and eventual nuclear envelope growth. Vesicle binding and fusion were assayed by light and electron microscopy. Addition of ATP and GTP to bound vesicles caused limited vesicle fusion, but enclosure of the chromatin was not observed. This result suggested that nondialyzable soluble components were required for nuclear vesicle fusion. GTP gamma S and guanylyl imidodiphosphate significantly inhibited vesicle fusion but had no effect on vesicle binding to chromatin. Preincubation of membranes with 1 mM GTP gamma S or GTP did not impair vesicle binding or fusion when assayed with fresh cytosol. However, preincubation of membranes with GTP gamma S plus cytosol caused irreversible inhibition of fusion. The soluble factor mediating the inhibition by GTP gamma S, which we named GTP-dependent soluble factor (GSF), was titratable and was depleted from cytosol by incubation with excess membranes plus GTP gamma S, suggesting a stoichiometric interaction between GSF and a membrane component in the presence of GTP gamma S. In preliminary experiments, cytosol depleted of GSF remained active for fusion of chromatin-bound vesicles, suggesting that GSF may not be required for the fusion reaction itself. We propose that GTP hydrolysis is required at a step before the fusion of nuclear vesicles.     

A lamin-independent pathway for nuclear envelope assembly

The nuclear envelope is composed of membranes, nuclear pores, and a nuclear lamina. Using a cell-free nuclear assembly extract derived from Xenopus eggs, we have investigated how these three components interact during nuclear assembly. We find that the Xenopus embryonic lamin protein LIII cannot bind directly to chromatin or membranes when each is present alone, but is readily incorporated into nuclei when both of the components are present together in an assembly extract. We find that depleting lamin LIII from an extract does not prevent formation of an envelope consisting of membranes and nuclear pores. However, these lamin-depleted envelopes are extremely fragile and fail to grow beyond a limited extent. This suggests that lamin assembly is not required during the initial steps of nuclear envelope formation, but is required for later growth and for maintaining the structural integrity of the envelope. We also present results showing that lamins may only be incorporated into nuclei after DNA has been encapsulated within an envelope and nuclear transport has been activated. With respect to nuclear function, our results show that the presence of a nuclear lamina is required for DNA synthesis to occur within assembled nuclei.     

Ran及其结合与调控蛋白在核膜装配过程中的调控作用

核膜在细胞周期中呈现高度的动态性：在细胞分裂的前中期,核膜崩解并分散到细胞质中;在细胞分裂的后期,核膜开始在染色体的表面重新装配,最终形成完整的核膜结构。近期的研究发现,Ran GTP酶、物质转运蛋白importinβ、内层核膜蛋白LBR（lamin B receptor）以及核孔复合体蛋白nucleoporins在核膜重建的过程中起关键性调控作用,并受到细胞周期调控因子p34cdc2激酶的调节。LBR是一个八次跨膜的膜蛋白,主要定位于内层核膜。在细胞分裂的早期,随着核膜崩解,LBR与核膜崩解而生成的小膜泡一起分散到细胞质中;在细胞分裂的后期,通过LBR与importinβ相互结合,含有LBR的膜泡被importinβ携带至染色质的表面参与核膜重建。目前已知p34cdc2激酶对LBR与importinβ介导的核膜重建起重要调控作用。Nucleoporins是核孔复合体主要组分。随核膜崩解,核孔复合体解聚成nucleoporins,分散到细胞质中,或结合到其他亚细胞成分上。细胞分裂后期,核孔复合体伴随核膜装配而组装。     

A role for nuclear lamins in nuclear envelope assembly.

The molecular interactions responsible for nuclear envelope assembly after mitosis are not well understood. In this study, we demonstrate that a peptide consisting of the COOH-terminal domain of Xenopus lamin B3 (LB3T) prevents nuclear envelope assembly in Xenopus interphase extracts. Specifically, LB3T inhibits chromatin decondensation and blocks the formation of both the nuclear lamina-pore complex and nuclear membranes. Under these conditions, some vesicles bind to the peripheral regions of the chromatin. These "nonfusogenic" vesicles lack lamin B3 (LB3) and do not bind LB3T; however, "fusogenic" vesicles containing LB3 can bind LB3T, which blocks their association with chromatin and, subsequently, nuclear membrane assembly. LB3T also binds to chromatin in the absence of interphase extract, but only in the presence of purified LB3. Additionally, we show that LB3T inhibits normal lamin polymerization in vitro. These findings suggest that lamin polymerization is required for both chromatin decondensation and the binding of nuclear membrane precursors during the early stages of normal nuclear envelope assembly.     

Plant nuclear envelope proteins

Compared to research in the animal field, the plant NE has been clearly under-investigated. The available data so far indicate similarities as well as striking differences that raise interesting questions about the function and evolution of the NE in different kingdoms. Despite a seemingly similar structure and organization of the NE, many of the proteins that are integral components of the animal NE appear to lack homologues in plant cells. The sequencing of the Arabidopsis genome has not led to the identification of homologues of animal NE components, but has indicated that the plant NE must have a distinct protein composition different from that found in metazoan cells. Besides providing a selective barrier between the nucleoplasm and the cytoplasm, the plant NE functions as a scaffold for chromatin but the scaffolding components are not identical to those found in animal cells. The NE comprises an MTOC in higher plant cells, a striking difference to the organization of microtubule nucleation in other eukaryotic cells. Nuclear pores are present in the plant NE, but identifiable orthologues of most animal and yeast nucleoporins are presently lacking. The transport pathway through the nuclear pores via the action of karyopherins and the Ran cycle is conserved in plant cells. Interestingly, RanGAP is sequestered to the NE in plant cells and animal cells, yet the targeting domains and mechanisms of attachment are different between the two kingdoms. At present, only a few proteins localized at the plant NE have been identified molecularly. Future research will have to expand the list of known protein components involved in building a functional plant NE.     

P446L-importin-beta inhibits nuclear envelope assembly by sequestering nuclear envelope assembly factors to the microtubules

The P446L mutant Drosophila importin-beta (P446L-imp-beta) has been reported to prohibit--in dominant negative fashion--nuclear envelope (NE) assembly. Along elucidating the mode of action of P446L-imp-beta we studied in vitro NE assembly on Sepharose beads. While Drosophila embryo extracts support NE assembly over Sepharose beads coated with Ran, NE assembly does not take place in extracts supplied with exogenous P446L-imp-beta. A NE also forms over importin-beta-coated beads. Surprisingly, when immobilized to Sepharose beads P446L-imp-beta as efficiently recruits NE vesicles as normal importin-beta. The discrepancy in behavior of cytoplasmic and bead-bound P446L-imp-beta appears to be related to icreased--as compared to normal importin-beta--microtubule (MT) binding ability of P446L-imp-beta. While wild-type importin-beta is able to bind MTs and the binding decreases upon RanGTP interaction, P446L-imp-beta cannot be removed from the MTs by RanGTP. P446L-imp-beta, like normal importin-beta, binds some types of the nucleoporins that have been known to be required for NE assembly at the end of mitosis. It appears that the inhibitory effect of P446L-imp-beta on NE assembly is caused by sequestering some of the nucleoporins required for NE assembly to the MTs.     

Minor capsid proteins of simian virus 40 are dispensable for nucleocapsid assembly and cell entry but are required for nuclear entry of the viral genome

We investigated the roles of simian virus 40 capsid proteins in the viral life cycle by analyzing point mutants in Vp1 and Vp2/3, as well as a deletion mutant lacking the Vp2/3 coding sequence. The Vp1 mutants (V243E and L245E) and the Vp2/3 mutants (F157E-I158E and P164R-G165E-G166R) were previously shown to be defective in Vp1-Vp2/3 interaction and to be noninfectious or poorly infectious, respectively. Here, we show that all these point mutants form stable particles following DNA transfection into cells. The Vp2/3-mutant particles contained very low levels of Vp2/3, whereas the Vp1 mutant particles contained no detectable Vp2/3. As expected, the deletion mutant also formed particles that were noninfectious. We further characterized the two Vp1 point mutants and the deletion mutant. All three mutant particles comprised Vp1 and histone-associated viral DNA, and all were able to enter cells. However, the mutant complexes failed to associate with host importins (owing to the loss of the Vp2/3 nuclear localization signal), and the mutant viral DNAs prematurely dissociated from the Vp1s, suggesting that the nucleocapsids did not enter the nucleus. Consistently, all three mutant particles failed to express large T antigen. Together, our results demonstrate unequivocally that Vp2/3 is dispensable for the formation of nucleocapsids. Further, the nucleocapsids' ability to enter cells implies that Vp1 contains the major determinants for cell attachment and entry. We propose that the major role of Vp2/3 in infectivity is to mediate the nuclear entry of viral DNA.     

Tyrosine kinase regulation of nuclear envelope assembly

We have described the co-localization of PLCγ and SFK1/7 in vivo and in vitro, and demonstrated their recruitment together to nuclei during membrane binding. We hypothesize that SFK1/7 is activated in response to GTP hydrolysis, phosphorylating and priming PLCγ for activation and thus producing the fusogen DAG during NE assembly. In addition to enhancing our understanding of the temporal and spatial mechanisms of NE formation, this pathway offers a novel link between protein regulation and lipid metabolism that has implications for general membrane fusion events.     

Early stages of the secretory pathway, but not endosomes, are required for Cvt vesicle and autophagosome assembly in Saccharomyces cerevisiae

The Cvt pathway is a biosynthetic transport route for a distinct subset of resident yeast vacuolar hydrolases, whereas macroautophagy is a nonspecific degradative mechanism that allows cell survival during starvation. Yet, these two vacuolar trafficking pathways share a number of identical molecular components and are morphologically very similar. For example, one of the hallmarks of both pathways is the formation of double-membrane cytosolic vesicles that sequester cargo before vacuolar delivery. The origin of the vesicle membrane has been controversial and various lines of evidence have implicated essentially all compartments of the endomembrane system. Despite the analogies between the Cvt pathway and autophagy, earlier work has suggested that the origin of the engulfing vesicle membranes is different; the endoplasmic reticulum is proposed to be required only for autophagy. In contrast, in this study we demonstrate that the endoplasmic reticulum and/or Golgi complex, but not endosomal compartments, play an important role for both yeast transport routes. Along these lines, we demonstrate that Berkeley bodies, a structure generated from the Golgi complex in sec7 cells, are immunolabeled with Atg8, a structural component of autophagosomes. Finally, we also show that none of the yeast t-SNAREs are located at the preautophagosomal structure, the presumed site of double-membrane vesicle formation. Based on our results, we propose two models for Cvt vesicle biogenesis.     

SNARE proteins are required for macroautophagy

Macroautophagy mediates the degradation of long-lived proteins and organelles via the de novo formation of double-membrane autophagosomes that sequester cytoplasm and deliver it to the vacuole/lysosome; however, relatively little is known about autophagosome biogenesis. Atg8, a phosphatidylethanolamine-conjugated protein, was previously proposed to function in autophagosome membrane expansion, based on the observation that it mediates liposome tethering and hemifusion in vitro. We show here that with physiological concentrations of phosphatidylethanolamine, Atg8 does not act as a fusogen. Rather, we provide evidence for the involvement of exocytic Q/t-SNAREs in autophagosome formation, acting in the recruitment of key autophagy components to the site of autophagosome formation, and in regulating the organization of Atg9 into tubulovesicular clusters. Additionally, we found that the endosomal Q/t-SNARE Tlg2 and the R/v-SNAREs Sec22 and Ykt6 interact with Sso1-Sec9, and are required for normal Atg9 transport. Thus, multiple SNARE-mediated fusion events are likely to be involved in autophagosome biogenesis.     

Relation between nuclear envelope and nuclear lamina in nuclear assembly in vitro

Xenopus laevis egg extracts cell-free nuclear assembly system was used as an experimental model to study the process of nuclear lamina assembly in nuclear reconstitution in vitro. The experimental results showed that lamin was involved in the nuclear assembly in vitro. The assembly of nuclear lamina was preceded by the assembly of nuclear matrix, and probably, inner nuclear matrix assembly provided the basis for nuclear lamina assembly. Inhibition of normal assembly of nuclear lamina, by preincubating egg extracts cell-free system with anti-lamin antibodies, resulted in abnormal assembly of nuclear envelope, suggesting that nuclear envelope assembly is closely associated with nuclear lamina assembly.     

Identification of cytosolic factors required for nuclear location sequence-mediated binding to the nuclear envelope

Nuclear protein import can be separated into two distinct steps: binding to the nuclear pore complex followed by translocation to the nuclear interior. A previously identified nuclear location sequence (NLS) receptor and a 97-kD protein purified from bovine erythrocytes reconstitute the binding step in a permeabilized cell assay. Binding to the envelope is specific for a functional SV-40 large T antigen NLS and is not ATP or temperature dependent. Modification of p97 with N- ethylmaleimide (NEM) decreases binding to the pore, but interestingly, NEM treatment of the NLS receptor does not. Nuclear envelope binding is inhibited by wheat germ agglutinin suggesting a possible mechanism for the inhibition of transport by the lectin.     

Yeast nuclear pore complex assembly defects determined by nuclear envelope reconstruction

Assembly of nuclear pore complexes (NPCs) is a critical yet poorly understood cellular function. One approach to studying NPC assembly is to identify yeast mutants defective in this process. This requires robust assays for NPC assembly that can be used for phenotypic analysis. We have previously reconstructed yeast nuclei from electron micrographs of serially sectioned cells to precisely determine the number of NPCs (Winey et al., 1997). Here we report the analysis of strains mutant in either of two nucleoporin-encoding genes, NIC96 (Zabel et al., 1996) and NUP192 (Kosova et al., 1999). Using conditional alleles of either gene, we have found that the NPC number falls significantly following shift to the restrictive temperature. We conclude that the drop in NPC number results from the failure to assemble new NPCs during cell divisions, leading to the dilution of NPCs that existed when the cells were shifted to the restrictive temperature. We are also able to document a subtle defect in NPC numbers in nup192-15 cells at their permissive temperature. The data presented here quantitatively demonstrate that NPC numbers fall in nic96-1 and nup192-15 strains upon shifting to the restrictive temperature, indicating that these gene products are required for NPC assembly.     

Kinesin-related proteins required for assembly of the mitotic spindle

We identified two new Saccharomyces cerevisiae kinesin-related genes, KIP1 and KIP2, using polymerase chain reaction primers corresponding to highly conserved regions of the kinesin motor domain. Both KIP proteins are expressed in vivo, but deletion mutations conferred no phenotype. Moreover, kip1 kip2 double mutants and a triple mutant with kinesin-related kar3 had no synthetic phenotype. Using a genetic screen for mutations that make KIP1 essential, we identified another gene, KSL2, which proved to be another kinesin-related gene, CIN8. KIP1 and CIN8 are functionally redundant: double mutants arrested in mitosis whereas the single mutants did not. The microtubule organizing centers of arrested cells were duplicated but unseparated, indicating that KIP1 or CIN8 is required for mitotic spindle assembly. Consistent with this role, KIP1 protein was found to colocalize with the mitotic spindle.     

Nuclear lamin proteins: domains required for nuclear targeting, assembly, and cell-cycle-regulated dynamics.

The nuclear lamin proteins assemble at the nuclear membrane into a highly dynamic network whose integrity is exquisitely controlled by the major cell cycle regulators. Although the actual cellular functions of the lamins remain elusive, the processes of targeted lamin assembly and phosphorylation-induced disassembly have come to light recently. These processes require functionally interacting lamin domains to execute targeting to the nucleus and the nuclear membranes, lamina assembly, and the disassembly response to cell-cycle-dependent phosphorylation.     

The extended loops of ribosomal proteins L4 and L22 are not required for ribosome assembly or L4-mediated autogenous control

Ribosomal proteins L4 and L22 both have a globular domain that sits on the surface of the large ribosomal subunit and an extended loop that penetrates its core. The tips of both loops contribute to the lining of the peptide exit tunnel and have been implicated in a gating mechanism that might regulate the exit of nascent peptides. Also, the extensions of L4 and L22 contact multiple domains of 23S rRNA, suggesting they might facilitate rRNA folding during ribosome assembly. To learn more about the roles of these extensions, we constructed derivatives of both proteins that lack most of their extended loops. Our analysis of ribosomes carrying L4 or L22 deletion proteins did not detect any significant difference in their sedimentation property or polysome distribution. Also, the role of L4 in autogenous control was not affected. We conclude that these extensions are not required for ribosome assembly or for L4-mediated autogenous control of the S10 operon.