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PubMed2007

Structural insight into the ESCRT-I/-II link and its role in MVB trafficking.

ESCRT (endosomal sorting complex required for transport) complexes orchestrate efficient sorting of ubiquitinated transmembrane receptors to lysosomes via multivesicular bodies (MVBs). Yeast ESCRT-I and ESCRT-II interact directly in vitro; however, this association is not detected in yeast cytosol. To gain understanding of the molecular mechanisms of this link, we have characterised the ESCRT-I/-II supercomplex and determined the crystal structure of its interface. The link is formed by the vacuolar protein sorting (Vps)28 C-terminus (ESCRT-I) binding with nanomolar affinity to the Vps36-NZF-N zinc-finger domain (ESCRT-II). A hydrophobic patch on the Vps28-CT four-helix bundle contacts the hydrophobic knuckles of Vps36-NZF-N. Mutation of the ESCRT-I/-II link results in a cargo-sorting defect in yeast. Interestingly, the two Vps36 NZF domains, NZF-N and NZF-C, despite having the same core fold, use distinct surfaces to bind ESCRT-I or ubiquitinated cargo. We also show that a new component of ESCRT-I, Mvb12 (YGR206W), engages ESCRT-I directly with nanomolar affinity to form a 1:1:1:1 heterotetramer. Mvb12 does not affect the affinity of ESCRT-I for ESCRT-II in vitro. Our data suggest a complex regulatory mechanism for the ESCRT-I/-II link in yeast.

Amino Acid Sequence↗
PubMed2006

Rb2/p130 and protein phosphatase 2A: key mediators of ovarian carcinoma cell growth suppression by all-trans retinoic acid.

Despite a number of attempts to improve treatment of ovarian cancer, it remains the most common cause of death from gynecological cancers. Thus, it is very important to identify more effective drugs for treatment and prevention of ovarian cancer. All-trans-retinoic acid (ATRA) has been shown to arrest the growth of ovarian carcinoma cells in G0/G1 and to significantly elevate levels of Rb2/p130 protein, a member of the retinoblastoma family of tumor suppressors. As ATRA treatment leads to a significant increase in the amount of Rb2/p130 protein but not mRNA, the elevated levels of Rb2/p130 protein is likely the result of increased stability. In studies to elucidate the mechanism by which ATRA alters Rb2/p130 stability in ovarian cancer cells, it was determined that PP2A, a serine/threonine phosphatase, binds and dephosphorylates Rb2/p130. Dephosphorylated Rb2/p130 exhibits decreased ubiquitination and thus is not degraded by the proteasome. The sites at which PP2A catalytic subunit (PP2Ac) interacts with Rb2/p130 have been localized to the NLS in the C-terminus of Rb2/p130. These sites are also involved in the interaction of Rb/p130 with importin beta and importin alpha, members of the nuclear transport machinery. It is known that importin alpha recognizes a NLS on a target protein and importin beta binds the nuclear pore complex. Moreover, it has been shown that the binding of importin alpha to NLS significantly decreases with phosphorylation of NLS. In ATRA-treated ovarian carcinoma cells, PP2A binds to Rb2/p130 and dephosphorylates the NLS of Rb2/p130 leading to the interaction of importin alpha with Rb2/p130. Importin beta then binds to the importin alpha-Rb2/p130 complex, leading to the translocation of the Rb2/p130 to the nucleus where it acts to arrest ovarian cancer cells in G1 and suppress proliferation.

Antineoplastic Agents↗
PubMed1980

Nucleocytoplasmic transport of RNA. The effect of 3'-deoxyadenosine triphosphate on RNA release from isolated nuclei.

The addition of 3'-deoxyadenosine (cordycepin) to cells in culture results in the inhibition of the appearance of mRNA in the cytoplasm through a mechanism thought to involve the inhibition of polyadenylate synthesis. I studied the effect of 3'-deoxyadenosine triphosphate, the physiologically active form of 3'-deoxyadenosine, on RNA release from isolated nuclei. Nuclei were isolated from baby-hamster kidney (BHK) fibroblasts that had been given a short pulse of radioactive uridine or adenosine in the presence of a low concentration of actinomycin D before harvest. RNA release from the isolated nuclei under the appropriate incubation conditions was time-, temperature- and ATP-dependent. 3'-Deoxyadenosine triphosphate inhibited RNA release from the isolated nuclei. However, RNA that was restricted to the nuclei during incubation with the drug could be chased out of the nuclei if the incubation medium was replaced with medium containing only ATP. The chased poly(A)+ (polyadenylated) RNA had shortened poly(A) tracts, indicating that poly(A)+ RNA with shortened poly(A) tracts can be transported out of the nucleus. An experiment was designed to test the effect of 3'-deoxyadenosine triphosphate on the release of poly(A)+ RNA at drug concentrations which caused 33 or 64% inhibition of RNA release. The release of poly(A)+ RNA and poly(A)- RNA (not polyadenylated) was equally inhibited by the drug. Thus, although 3'-deoxyadenosine triphosphate does inhibit release of RNA from the nucleus, it would appear that the drug does so through a mechanism independent of the inhibition of polyadenylation. The process that is inhibited must be one that is common to both poly(A)+ and poly(A)- RNA. The possibility that 3'-deoxyadenosine triphosphate inhibits a reaction at the nuclear membrane or nuclear pore complex is considered.

Animals↗
PubMed1999

Visualization of biphasic Ca2+ diffusion from cytosol to nucleus in contracting adult rat cardiac myocytes with an ultra-fast confocal imaging system.

In contracting cardiac myocytes, the rapid changes in cytosolic and nuclear Ca2+ make it difficult to determine whether the nuclear Ca2+ transient is caused by diffusion from the cytosol or by Ca2+ release channels on the inner nuclear membrane, or both. The propagation mechanism in the nucleoplasm also remains unknown. We have developed an ultra-fast Nipkow confocal imaging system able to acquire two-dimensional images at approximately 4 ms/full frame speed and employed it to analyze Ca2+ waves and the dynamics of the cytosolic and nuclear Ca2+ transients after electrical stimulation of cardiac myocytes. The pattern of nuclear Ca2+ upon stimulation was well described by a mathematical model of Ca2+ diffusion across the nuclear envelope. No evidence of Ca2+ release from perinuclear Ca2+ stores was obtained. The Ca2+ diffusion constant appeared to change during contraction, with essentially free diffusion of Ca2+ through nuclear pore complexes at low cytosolic Ca2+ and partially restricted diffusion at high cytosolic Ca2+. The Ca2+ in the nucleoplasm propagated by diffusion and no Ca2+ release phenomena were seen in the nucleus.

Animals↗
PubMed2001

Kinetics of protein import into isolated Xenopus oocyte nuclei.

An in vitro assay for nucleocytoplasmic transport was established in which signal-dependent protein import is reproduced faithfully by isolated purified nuclei. The assay permits the precise quantification of import kinetics and the discrimination between translocation through the nuclear envelope and intranuclear transport. Nuclei were manually isolated from Xenopus oocytes and after manual purification incubated with a medium containing a green fluorescent transport substrate, karyopherins alpha2 and beta1, a red fluorescent control substrate, an energy mix and, for keeping an osmotic balance, 20% (wt/vol) BSA. Import of transport substrates into the nucleus and exclusion of the control substrate were monitored simultaneously by two-color confocal microscopy. Two widely differing import substrates were used: the recombinant protein P4K [480 kDa, four nuclear localization sequences (NLSs) per P4K tetramer], and NLS-BSA (90 kDa, 15 NLSs). The measurements suggested that import, at the specific conditions used in this study, consisted of two consecutive processes: (i) the rapid equilibration of the concentration difference across the nuclear envelope, a process involving binding and translocation of substrate by the nuclear pore complex, and (ii) the dissipation of the intranuclear concentration difference by diffusion.

Animals↗
PubMed2001

Multiple vesiculoviral matrix proteins inhibit both nuclear export and import.

The matrix (M) protein of vesicular stomatitis virus inhibits both nuclear import and export. Here, we demonstrate that this inhibitory property is conserved between the M proteins from two other vesiculoviruses, chandipura virus and spring viremia carp virus. All three M proteins completely block nuclear transport of spliced mRNA, small nuclear RNAs, and small nuclear ribonucleoproteins and slow the nuclear transport of many other cargoes. In all cases where transport was merely slowed by the M proteins, the chandipura virus M protein had the strongest inhibitory activity. When expressed in transfected HeLa cells, active M proteins displayed prominent association with the nuclear rim. Moreover, mutation of a conserved methionine abolished both the inhibitory activity and efficient targeting of the M proteins to the nuclear rim. We propose that all of the vesiculoviral M proteins associate with the same nuclear target, which is likely to be a component of the nuclear pore complex.

Active Transport, Cell Nucleus↗
PubMed1997

Anti-idiotype RNA selected with an anti-nuclear export signal antibody is actively transported in oocytes and inhibits Rev- and cap-dependent RNA export.

The anti-idiotype approach is based on the assumption that an antibody specific for a receptor-binding domain of a ligand could be structurally related to the receptor. Therefore, a structural mimic of a receptor-binding domain, selected with an anti-ligand antibody, might be a functional substrate for the receptor. This hypothesis was addressed here by generating antibodies recognizing the Rev-nuclear export signal (NES). A functional NES is required for active export, presumably by interacting directly or indirectly with the nuclear pore complex. Anti-NES antibodies were used to isolate RNA mimics of the NES peptide from combinatorial RNA libraries. The RNA-mimics are exported actively, block Rev-dependent export of a reporter RNA, and inhibit cap-dependent U1 snRNA export in Xenopus oocytes, properties previously reported for NES-peptide conjugates.

Animals↗
PubMed2002

Identification of a C-terminal region that is required for the nuclear translocation of ERK2 by passive diffusion.

Extracellular signal-regulated kinase 2 (ERK2) is located in the cytoplasm of resting cells and translocates into the nucleus upon extracellular stimuli by active transport of a dimer. Passive transport of an ERK2 monomer through the nuclear pore is also reported to coexist. We attempted to characterize the cytoplasmic retention and nuclear translocation of fusion proteins between deletion and site-directed mutants of ERK2 and green fluorescent protein (GFP). The overexpressed ERK2-GFP fusion protein is usually localized to both the cytoplasm and the nucleus unless a cytoplasmic anchoring protein is coexpressed. Deletion of 45 residues, but not 43 residues, from the C terminus of ERK2 prevented the nuclear distribution of the ERK2-GFP fusion protein. Substitution of a part of residues 299-313 to alanine residues also prevented the nuclear distribution of the ERK2-GFP fusion protein without abrogation of its nuclear active transport. These observations may indicate that the passive diffusion of ERK2 into the nucleus is not simple diffusion but includes a specific interaction process between residues 299-313 and the nuclear pore complex and that this interaction is not required for the active transport. We also showed that substitution of Tyr(314) to alanine residue abrogated the cytoplasmic retention of the ERK2-GFP fusion protein by PTP-SL but not by MEK1.

Active Transport, Cell Nucleus↗
PubMed2003

Oligomerization-induced modulation of TPR-MET tyrosine kinase activity.

Phosphorylation, although necessary, may not be sufficient to fully activate many receptor tyrosine kinases (RTKs). Oligomerization-induced conformational changes may be necessary to modulate the kinetic properties of RTKs and render them fully functional. To investigate this regulatory mechanism, recombinant TPR-MET, a functionally active oncoprotein derivative of the RTK c-MET, has been expressed and purified for quantitative enzymatic analysis. This naturally occurring oncoprotein contains the cytoplasmic domain of c-MET fused to a coiled coil motif from the nuclear pore complex (TPR). cytoMET, the monomeric analog of TPR-MET, has also been expressed and purified for comparative enzymatic analysis. ATP and peptide substrates have been kinetically characterized for both TPR-MET and cytoMET. Significantly, phosphorylated TPR-MET has smaller Km values for ATP (Km,ATP) and peptide substrates (Km,peptide) and a larger kcat relative to phosphorylated cytoMET. This provides the first direct evidence that receptor oligomerization and not simply activation loop phosphorylation modulates RTK enzymatic activity. The ATP dissociation constants (Kd,ATP) for the two enzymes also displayed significant differences. In contrast, the KI values for the ATP competitive inhibitor staurosporin are similar for the two phosphorylated enzymes. These results suggest that much of the oligomerization-induced kinetic changes occur with respect to peptide substrate binding or catalytic efficiency. The possibility that oligomerization-induced conformational changes occur within the cytoplasmic domain of receptor tyrosine kinases has significant implications for structure-based design of RTK inhibitors and the development of a detailed mechanistic model of RTK activation.

Adenosine Triphosphate↗
PubMed1986

Determination of the intracellular state of soluble macromolecules by gel filtration in vivo in the cytoplasm of amphibian oocytes.

A method to examine the diffusible state and the sizes of major cytoplasmic proteins in a living cell is described. Small (40-300 microns) commercially available gel filtration beads of a broad range of Mr exclusion limits were microsurgically implanted into the cytoplasm of oocytes of the frog, Xenopus laevis, usually after metabolic labeling of oocyte proteins with [35S]methionine. After equilibration in vivo for several hours, the appearance of the implanted cells, notably the bead-cytoplasm boundary, was examined by light and electron microscopy of sections and found to be unaffected. After incubation the beads were isolated, briefly rinsed, and their protein contents examined by one- or two-dimensional gel electrophoresis. We show that diffusible proteins can be identified by their inclusion in the pores of the gel filtration beads used and that their approximate sizes can be estimated from the size exclusion values of the specific materials used. The application of this method to important cell biological questions is demonstrated by showing that several "karyophobic proteins," i.e., proteins of the cytosolic fraction which accumulate in the cytoplasm in vivo, are indeed diffusible in the living oocyte and appear with sizes similar to those determined in vitro. This indicates that the nucleo-cytoplasmic distribution of certain diffusible proteins is governed, in addition to size exclusion at nuclear pore complexes and karyophilic "signals," by other, as yet unknown forces. Some possible applications of this method of gel filtration in vivo are discussed.

Animals↗
PubMed1986

A fractionated cell-free system for analysis of prophase nuclear disassembly.

We describe a cell-free system in which a postribosomal supernatant (s140) from metaphase Chinese hamster ovary (CHO) cells induces prophase-like changes in isolated CHO cell nuclei, including chromatin condensation, and nuclear envelope and lamina disassembly. These events are strongly promoted by gamma-S-ATP and an ATP-regenerating system, and do not take place with an s140 derived from G2-phase cells. The metaphase cell s140 also induces disassembly of an isolated nuclear lamina fraction that is depleted of membranes, chromatin, and nuclear pore complexes. Disassembly of the isolated lamina is accompanied by phosphorylation of the major lamina proteins (lamins A, B, and C) to levels characteristic of metaphase cells. Kinetic analysis of lamina depolymerization indicates that cooperativity may be involved in this process. The biochemical properties of in vitro lamina disassembly suggest that the activity that depolymerizes the lamina during mitosis is soluble in metaphase cells, and support the notion that this activity is a lamin protein kinase.

Animals↗
PubMed1997

Microtubule-mediated transport of incoming herpes simplex virus 1 capsids to the nucleus.

Herpes simplex virus 1 fuses with the plasma membrane of a host cell, and the incoming capsids are efficiently and rapidly transported across the cytosol to the nuclear pore complexes, where the viral DNA genomes are released into the nucleoplasm. Using biochemical assays, immunofluorescence, and immunoelectron microscopy in the presence and absence of microtubule depolymerizing agents, it was shown that the cytosolic capsid transport in Vero cells was mediated by microtubules. Antibody labeling revealed the attachment of dynein, a minus end-directed, microtubule-dependent motor, to the viral capsids. We propose that the incoming capsids bind to microtubules and use dynein to propel them from the cell periphery to the nucleus.

Actin Cytoskeleton↗
PubMed2003

Traffic-independent function of the Sar1p/COPII machinery in proteasomal sorting of the cystic fibrosis transmembrane conductance regulator.

Newly synthesized proteins that do not fold correctly in the ER are targeted for ER-associated protein degradation (ERAD) through distinct sorting mechanisms; soluble ERAD substrates require ER-Golgi transport and retrieval for degradation, whereas transmembrane ERAD substrates are retained in the ER. Retained transmembrane proteins are often sequestered into specialized ER subdomains, but the relevance of such sequestration to proteasomal degradation has not been explored. We used the yeast Saccharomyces cerevisiae and a model ERAD substrate, the cystic fibrosis transmembrane conductance regulator (CFTR), to explore whether CFTR is sequestered before degradation, to identify the molecular machinery regulating sequestration, and to analyze the relationship between sequestration and degradation. We report that CFTR is sequestered into ER subdomains containing the chaperone Kar2p, and that sequestration and CFTR degradation are disrupted in sec12ts strain (mutant in guanine-nucleotide exchange factor for Sar1p), sec13ts strain (mutant in the Sec13p component of COPII), and sec23ts strain (mutant in the Sec23p component of COPII) grown at restrictive temperature. The function of the Sar1p/COPII machinery in CFTR sequestration and degradation is independent of its role in ER-Golgi traffic. We propose that Sar1p/COPII-mediated sorting of CFTR into ER subdomains is essential for its entry into the proteasomal degradation pathway. These findings reveal a new aspect of the degradative mechanism, and suggest functional crosstalk between the secretory and the degradative pathways.

COP-Coated Vesicles↗
PubMed2003

Function of the p97-Ufd1-Npl4 complex in retrotranslocation from the ER to the cytosol: dual recognition of nonubiquitinated polypeptide segments and polyubiquitin chains.

A member of the family of ATPases associated with diverse cellular activities, called p97 in mammals and Cdc48 in yeast, associates with the cofactor Ufd1-Npl4 to move polyubiquitinated polypeptides from the endoplasmic reticulum (ER) membrane into the cytosol for their subsequent degradation by the proteasome. Here, we have studied the mechanism by which the p97-Ufd1-Npl4 complex functions in this retrotranslocation pathway. Substrate binding occurs when the first ATPase domain of p97 (D1 domain) is in its nucleotide-bound state, an interaction that also requires an association of p97 with the membrane through its NH2-terminal domain. The two ATPase domains (D1 and D2) of p97 appear to alternate in ATP hydrolysis, which is essential for the movement of polypeptides from the ER membrane into the cytosol. The ATPase itself can interact with nonmodified polypeptide substrates as they emerge from the ER membrane. Polyubiquitin chains linked by lysine 48 are recognized in a synergistic manner by both p97 and an evolutionarily conserved ubiquitin-binding site at the NH2 terminus of Ufd1. We propose a dual recognition model in which the ATPase complex binds both a nonmodified segment of the substrate and the attached polyubiquitin chain; polyubiquitin binding may activate the ATPase p97 to pull the polypeptide substrate out of the membrane.

Adenosine Triphosphatases↗
PubMed1972

Time sequence of nuclear pore formation in phytohemagglutinin-stimulated lymphocytes and in HeLa cells during the cell cycle.

The time sequence of nuclear pore frequency changes was determined for phytohemagglutinin (PHA)-stimulated human lymphocytes and for HeLa S-3 cells during the cell cycle. The number of nuclear pores/nucleus was calculated from the experimentally determined values of nuclear pores/micro(2) and the nuclear surface. In the lymphocyte system the number of pores/nucleus approximately doubles during the 48 hr after PHA stimulation. The increase in pore frequency is biphasic and the first increase seems to be related to an increase in the rate of protein synthesis. The second increase in pores/nucleus appears to be correlated with the onset of DNA synthesis. In the HeLa cell system, we could also observe a biphasic change in pore formation. Nuclear pores are formed at the highest rate during the first hour after mitosis. A second increase in the rate of pore formation corresponds in time with an increase in the rate of nuclear acidic protein synthesis shortly before S phase. The total number of nuclear pores in HeLa cells doubles from approximately 2000 in G(1) to approximately 4000 at the end of the cell cycle. The doubling of the nuclear volume and the number of nuclear pores might be correlated to the doubling of DNA content. Another correspondence with the nuclear pore number in S phase is found in the number of simultaneously replicating replication sites. This number may be fortuitous but leads to the rather speculative possibility that the nuclear pore might be the site of initiation and/or replication of DNA as well as the site of nucleocytoplasmic exchange. That is, the nuclear pore complex may have multiple functions.

Cell Nucleus↗
PubMed1974

Reversible, thermotropic alteration of nuclear membrane stucture and nucleocytoplasmic RNA transport in Tetrahymena.

We examine the effect of cooling upon the freeze-etch ultrastructure of nuclear membranes, as well as upon nucleocytoplasmic RNA transport in the unicellular eukaryote Tetrahymena pyriformis. Chilling produces smooth, particle-free areas on both faces of the two freeze-fractured macronuclear membranes. Upon return to optimum growth temperature the membrane-associated particles revert to their normal uniform distribution and the smooth areas disappear. Chilling lowers the incorporation of [(14)C]uridine into whole cells and their cytoplasmic RNA. Cooling from the optimum growth temperature of 28 degrees to 18 degrees C (or above) decreases [(14)C]uridine incorporation into cells more than into their cytoplasmic RNA; chilling to below 18 degrees C but above 10 degrees C causes the reverse. [(14)C]Uridine incorporation into whole cells and their cytoplasmic RNA reflects overall RNA synthesis and nucleocytoplasmic RNA transport, respectively. RNA transport decreases strongly between 20 degrees and 16 degrees C, which is also the temperature range where morphologically detectable nuclear membrane transitions occur. This suggests that the nuclear envelope limits the rate of nucleocytoplasmic RNA transport at low temperatures. We hypothesize that a thermotropic lipid phase transition switches nuclear pore complexes from an "open" to a "closed" state with respect to nucleocytoplasmic RNA transport.

Animals↗
PubMed1977

Reversibly contractile nuclear matrix. Its isolation, structure, and composition.

From Tetrahymena macronuclei we have isolated a reversibly contractile nucleo-skeleton, i.e., an "expanded" nuclear matrix which reversibly contracts when the total concentration of the bivalent cations, Ca and Mg (3:2), is decreased to 5 mM or increased to 125 mM. During contraction the average diameter of the expanded matrix becomes reduced by about 24%; this corresponds to a volume contraction of about 55%. The reversible contraction of the nuclear matrix does not depend on ATP and cannot be inhibited by salygran. The expanded matrix is obtained by removing carefully from the macronuclei 89.7% of the phospholipid, 99.6% of the DNA, 98.5% of the RNA, and 74.8% of the protein by treatment with Triton X-100 and digestion with DNase and RNase followed by an extraction with 2 M NaCl. Electron microscopy reveals, within the expanded matrix, residual equivalents to the structures characteristic for macronuclei: (a) a residual nuclear envelope with nuclear pore complexes; (b) residual nucleoli at the periphery; (c) a fibrillar internal network. The expanded matrix is essentially composed of proteins (96.2%) and traces of DNA (0.8%), RNA (0.5%), phospholipid (1.6%), and carbohydrates (0.9%). The last, which have been determined by gas chromatography, contain glucose, mannose, and an unidentified sugar in the ratio 1:5.4:5.7. The ratio of acidic to basic amino acids of the expanded matrix is 1.55. Sodium dodecyl sulfate (SDS) gel electrophoresis reveals a predominant protein with a mol wt of 18,000 which is apparently involved in the reversible contractile process. The mechanism of this reversible contraction of the expanded matrix remains to be elucidated, but it differs both from actin-myosin contraction systems and from the contractile spasmoneme system in vorticellids.

Calcium↗
PubMed2001

HRD4/NPL4 is required for the proteasomal processing of ubiquitinated ER proteins.

We isolated a temperature-sensitive mutant, hrd4-1, deficient in ER-associated degradation (ERAD). The HRD4 gene was identical to NPL4, a gene previously implicated in nuclear transport. Using a diverse set of substrates and direct ubiquitination assays, our analysis revealed that HRD4/NPL4 is required for a poorly characterized step in ERAD after ubiquitination of target proteins but before their recognition by the 26S proteasome. Our data indicate that this lack of proteasomal processing of ubiquitinated proteins constitutes the primary defect in hrd4/npl4 mutant cells and explains the diverse set of hrd4/npl4 phenotypes. We also found that each member of the Cdc48p-Ufd1p-Npl4p complex is individually required for ERAD.

Active Transport, Cell Nucleus↗