Characterization of a messenger RNA transport protein

A cytoplasmic protein which facilitates the energy-dependent transport of mRNA from isolated nuclei to a specified medium has been further characterized, since it could have relevance to the mechanism of mRNA nucleo-cytoplasmic transport in vivo. This protein is now shown, by cDNA hybridization analysis using appropriate recombinant probes, to be obligatory for the transport of alpha 2u-globulin and albumin mRNA from male rat liver nuclei. It is concentrated in the cytoplasm. When isolated under conditions where they retain nuclear proteins, the nuclei contain less than 2% of the total mRNA transport activity. Approx. 20% is recovered in the cytosol, while the rest (80%) copurifies with the messenger ribonucleoproteins in the polyribosome fraction. The protein is eluted from the poly A-messenger ribonucleoproteins between 0.25 and 0.50 M NaCl. The activities of the cytosolic- and messenger ribonucleoprotein-derived transport proteins were mutually additive below saturation of the transport system. Further, the activities of both fractions were increased when they were fortified with the catalytic subunit of the cAMP-dependent protein kinase in the presence of ATP. On the other hand, protein kinase-induced thiophosphorylation of the protein with ATP[S] decreased transport activity. The molecular weight of the transport protein from either cell compartment as judged by molecular sieving is approx. 35,000. It has now been purified 2000-fold and requires manganese ions and serum albumin for stabilization of activity. The highly purified transport factor from the cytosol is tentatively assigned a molecular weight of 32,000 by SDS-polyacrylamide gel electrophoresis.     

Control of eIF4E cellular localization by eIF4E-binding proteins, 4E-BPs

Eukaryotic initiation factor (eIF) 4E, the mRNA 5'-cap-binding protein, mediates the association of eIF4F with the mRNA 5'-cap structure to stimulate cap-dependent translation initiation in the cytoplasm. The assembly of eIF4E into the eIF4F complex is negatively regulated through a family of repressor proteins, called the eIF4E-binding proteins (4E-BPs). eIF4E is also present in the nucleus, where it is thought to stimulate nuclear-cytoplasmic transport of certain mRNAs. eIF4E is transported to the nucleus via its interaction with 4E-T (4E-transporter), but it is unclear how it is retained in the nucleus. Here we show that a sizable fraction (approximately 30%) of 4E-BP1 is localized to the nucleus, where it binds eIF4E. In mouse embryo fibroblasts (MEFs) subjected to serum starvation and/or rapamycin treatment, nuclear 4E-BPs sequester eIF4E in the nucleus. A dramatic loss of nuclear 4E-BP1 occurs in c-Ha-Ras-expressing MEFs, which fail to show starvation-induced nuclear accumulation of eIF4E. Therefore, 4E-BP1 is a regulator of eIF4E cellular localization.     

蛋白质入核转运的机制和研究进展

细胞核膜是由外膜和内膜组成的磷脂双分子层结构,同时镶嵌一些核孔复合体（NPC）.核孔复合体是胞浆和胞核之间主动和被动转运的生理屏障.核内功能蛋白在胞浆内合成后通过核孔复合体进入胞核,这个过程除了需要NPC上核孔蛋白、胞浆内核转运受体和RanGTP等蛋白的参与外, 货物蛋白本身的结构特征在其入核转运过程中亦发挥重要作用.本文着重就蛋白入核转运的机制及近年来取得的相关进展进行综述.     

Intracellular distribution of the U1A protein depends on active transport and nuclear binding to U1 snRNA.

Nuclear transport of the U1 snRNP-specific protein U1A has been examined. U1A moves to the nucleus by an active process which is independent of interaction with U1 snRNA. Nuclear localization requires an unusually large sequence element situated between amino acids 94 and 204 of the protein. U1A transport is not unidirectional. The protein shuttles between nucleus and cytoplasm. At equilibrium, the concentration of the protein in the nucleus and cytoplasm is not, however, determined solely by transport rates, but can be perturbed by introducing RNA sequences that can specifically bind U1A in either the nuclear or cytoplasmic compartment. Thus, U1A represents a novel class of protein which shuttles between cytoplasm and nucleus and whose intracellular distribution can be altered by the number of free binding sites for the protein present in the cytoplasm or the nucleus.     

THE EFFECT OF PROTEIN SYNTHESIS INHIBITION ON THE ENTRY OF MESSENGER RNA INTO THE CYTOPLASM OF SEA URCHIN EMBRYOS

Emetine is a potent inhibitor of protein synthesis in sea urchin embryos. At a concentration of the drug that rapidly inhibits protein synthesis in blastulae by 95%, uridine incorporation into RNA continues for more than 1 hr and presumptive histone messenger RNA is synthesized and transported into the cytoplasm where it is apparently associated with polyribosomes. Possible explanations of this result and its implications for the "informasome" theory of messenger transport in embryonic cells are discussed.     

YAP nuclear‐cytoplasmic translocation is regulated by mechanical signaling,protein modification,and metabolism

Nuclear‐cytoplasmic transport is necessary for the biological function of nuclear proteins. The mechanism underlying this process is very complex and has been a subject of intense research. Yes‐associated protein (YAP), a Hippo signaling pathway effector, localizes to both the cytoplasm and the nucleus and can influence cell proliferation, stem cell status, and tissue homeostasis. Recent studies have focused on the significance of YAP distribution between the nucleus and the cytoplasm in disease, but it remains unclear how this dynamic process is regulated. In this review, we discuss YAP nuclear‐cytoplasmic transport under different physiological and pathological conditions in terms of mechanical signaling, protein modification, and metabolism. Understanding the mechanisms underlying nuclear‐cytoplasmic YAP transport mechanism under different physiological and pathological conditions may help identify important targets for disease treatment.     

A new role of ran GTPase.

Ran is a G protein similar to Ras, but it has no membrane binding site. RanGEF, RCC1, is on chromatin and RanGAP, RanGAP1/Rna1p is in cytoplasm. Ran, thus, shuttles between the nucleus and the cytoplasm to complete its GTPase cycle, carrying out nucleocytoplasmic transport of macromolecules. A majority of Ran binding proteins, thus far found, are required for this process. A recently found novel Ran-binding protein, RanBPM, however, is localized in the centrosome. Subsequently, four groups reported that RanGTP, but not RanGDP, can induce microtubule self-organization in Xenopus egg extracts where no nuclear membrane is present. Thus, Ran is suggested to have a new role beyond the nucleocytoplasmic transport of macromolecules. In both microtubule assembly and nucleocytoplasmic transport, chromosomal localization of RCC1 is important to carry out the functions of RanGTPase. In this regard, a future intriguing question is how RCC1 interacts with chromatin DNA.     

A membrane glycoprotein, Sec12p, required for protein transport from the endoplasmic reticulum to the Golgi apparatus in yeast

SEC12, a gene that is required for secretory, membrane, and vacuolar proteins to be transported from the endoplasmic reticulum to the Golgi apparatus, has been cloned from a genomic library by complementation of a sec12 ts mutation. Genetic analysis has shown that the cloned gene integrates at the SEC12 locus and that a null mutation at the locus is lethal. The DNA sequence predicts a protein of 471 amino acids containing a hydrophobic stretch of 19 amino acids near the COOH terminus. To characterize the gene product (Sec12p) in detail, a lacZ-SEC12 gene fusion has been constructed and a polyclonal antibody raised against the hybrid protein. The antibody recognizes Sec12p as a approximately 70-kD protein that sediments in a mixed membrane fraction that includes endoplasmic reticulum. Sec12p is not removed from the membrane fraction by treatment at high pH and high salt and is not degraded by exogenous protease unless detergent is present. Glycosylation of Sec12p during biogenesis is indicated by an electrophoretic mobility shift of the protein that is influenced by tunicamycin and by imposition of an independent secretory pathway block. We suggest that Sec12p is an integral membrane glycoprotein with a prominent domain that faces the cytoplasm where it functions to promote protein transport to the Golgi apparatus. In the process of transport, Sec12p itself may migrate to the Golgi apparatus and function in subsequent transport events.     

Transport of haemin across the cytoplasmic membrane through a haemin-specific periplasmic binding-protein-dependent transport system in Yersinia enterocolitica

The Yersinia enterocolitica O:8 periplasmic binding-protein-dependent transport (PBT) system for haemin was cloned and characterized. It consisted of four proteins: the periplasmic haemin-binding protein HemT, the haemin permease protein HemU, the ATP-binding hydrophilic protein HemV and the putative haemin-degrading protein HemS. Y. enterocolitica strains mutated in hemU or hemV genes were unable to use haemin as an iron source whereas those mutated in the hemT gene were able to use haemin as an iron source. As Escherichia coli strains expressing only the haemin outer membrane receptor protein HemR from Y. enterocolitica were capable of using haemin as an iron source the existence of an E. coli K-12 haemin-specific PBT system is postulated. The first gene in the Y. enterocolitica haemin-specific PBT system encoded a protein, HemS, which is probably involved in the degradation of haemin in the cytoplasm. The presence of the hemS gene was necessary to prevent haemin toxicity in E. coli strains that accumulate large amounts of haemin in the cytoplasm. We propose a model of haemin utilization in Y. enterocolitica in which HemT, HemU and HemV proteins transport haemin into the cytoplasm where it is degraded by HemS thereby liberating the iron.     

Use of External, Biosynthetic, and Organellar Arginine by Neurospora

The fate of very low amounts of (14)C-arginine derived from the medium or from biosynthesis was studied in Neurospora cells grown in minimal medium. In both cases, the label enters the cytoplasm, where it is very briefly used with high efficiency for protein synthesis without mixing with the bulk of the large, endogenous pool of (12)C-arginine. The soluble (14)C-arginine which is not used for protein synthesis is sequestered in a vesicle with the bulk of the endogenous arginine pool. After this time, it is selectively excluded from use in protein synthesis except by exchange with cytoplasmic arginine. The data suggest that in vivo, the non-organellar cytoplasm contains less than 5% of the soluble, cellular arginine. The cellular organization of Neurospora described here also prevents the catabolism of arginine. Our results are discussed in relation to previous work on amino acid pools of other eukaryotic systems.     

Identification of the human mitochondrial oxodicarboxylate carrier. Bacterial expression, reconstitution, functional characterization, tissue distribution, and chromosomal location

In Saccharomyces cerevisiae, the genes ODC1 and ODC2 encode isoforms of the oxodicarboxylate carrier. They both transport C5-C7 oxodicarboxylates across the inner membranes of mitochondria and are members of the family of mitochondrial carrier proteins. Orthologs are encoded in the genomes of Caenorhabditis elegans and Drosophila melanogaster, and a human expressed sequence tag (EST) encodes part of a closely related protein. Information from the EST has been used to complete the human cDNA sequence. This sequence has been used to map the gene to chromosome 14q11.2 and to show that the gene is expressed in all tissues that were examined. The human protein was produced by overexpression in Escherichia coli, purified, and reconstituted into phospholipid vesicles. It has similar transport characteristics to the yeast oxodicarboxylate carrier proteins (ODCs). Both the human and yeast ODCs catalyzed the transport of the oxodicarboxylates 2-oxoadipate and 2-oxoglutarate by a counter-exchange mechanism. Adipate, glutarate, and to a lesser extent, pimelate, 2-oxopimelate, 2-aminoadipate, oxaloacetate, and citrate were also transported by the human ODC. The main differences between the human and yeast ODCs are that 2-aminoadipate is transported by the former but not by the latter, whereas malate is transported by the yeast ODCs but not by the human ortholog. In mammals, 2-oxoadipate is a common intermediate in the catabolism of lysine, tryptophan, and hydroxylysine. It is transported from the cytoplasm into mitochondria where it is converted into acetyl-CoA. Defects in human ODC are likely to be a cause of 2-oxoadipate acidemia, an inborn error of metabolism of lysine, tryptophan, and hydroxylysine.     

The nuclear pore complex, nuclear transport, and apoptosis

The nuclear pore complex (NPC) is the sole gateway between the nucleus and the cytoplasm of interphase eukaryotic cells, and it mediates all trafficking between these 2 cellular compartments. As such, the NPC and nuclear transport play central roles in translocating death signals from the cell membrane to the nucleus where they initiate biochemical and morphological changes occurring during apoptosis. Recent findings suggest that the correlation between the NPC, nuclear transport, and apoptosis goes beyond the simple fact that NPCs mediate nuclear transport of key players involved in the cell death program. In this context, the accessibility of key regulators of apoptosis appears to be highly modulated by nuclear transport (e.g., impaired nuclear import might be an apoptotic trigger). In this review, recent findings concerning the unexpected tight link between NPCs, nuclear transport, and apoptosis will be presented and critically discussed.     

NSP-interacting GTPase: A cytosolic protein as cofactor for nuclear shuttle proteins

Despite the significant progress in the identification of essential components of the nuclear transport machinery, some events of this process are still unclear. Particularly, functional information about the release of nuclear-exported macromolecules at the cytoplasmic side of the nuclear pore complex and their subsequent trans-cytoplasmic movement is lacking. Recently, we identified a cytoplasmic GTPase, designated NIG (NSP-interacting GTPase), which may play a relevant role in these processes. NIG interacts in vivo with the geminivirus NSP and promotes the translocation of the viral protein from the nucleus to the cytoplasm where it is redirected to the cell surface to interact with the viral movement protein, MP. Here we position the NIG function into the mechanistic model for the intracellular trafficking of viral DNA and discuss the putative role of NIG in general cellular nucleocytoplasmic transport of nucleic acid-protein complexes.Key words: geminivirus, NIG, NSP, nucleocytoplasmic trafficking, transport activity     

Protein transport: A selective membrane mechanism

Proteins are selectively sequestered by a number of cell types. However, only in oocytes is the process sufficiently aggravated and specific to be readily studied. In these cells certain serum proteins are taken up in proportions different from those found in the serum. In vitro incubations of hormonally stimulated and synchronous mosquito oocytes show that the only protein capable of initiating the transport process is the female specific yolk protein. Heterologous proteins such as IgG, bovine serum albumin, cytochrome C, and ferritin are inactive. The female specific protein is a phosphoglycolipoprotein. It is synthesized in the fat body, a liver analog in the insect, and passed into the serum before being transported into the oocytes. Preliminary kinetic analysis shows the uptake process to be specific with an apparent K_m of about 10^?7 M. Glycolytic inhibitors stop protein uptake. The receptor-mediated binding steps in the transport process are most easily studied in the chicken because of the enormous amount of oocyte membrane available from a given oocyte and because up to 1 gm of protein is normally transported per day per oocyte. IgG and the hen specific phosvitin lipovitellin are two of the physiologically important proteins that are transported intact into the chicken oocytes. The uptake appears selective as shown by studies with iodinated proteins. Ferritin conjugated to IgG is shown by electron microscopy to bind to isolated plasma membranes only where coated pits have formed, whereas ferritin alone is not seen localized on any membrane surface. These very specialized regions of the membrane are similar to micropinocytotic pits but, in addition, possess on their cytoplasmic side dense ridges that form the coat. Transport involves binding to the coated pits, the pinching off of the pits, and the subsequent movement of the coated vesicles in the cytoplasm.