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The Ran-GTPase cycle and certain nucleoporins necessary for export from the nucleus of the small ribosomal subunit are discussed. Information is based on a study using the cytoplasmic localization of the 5' ITS1 fragment as indicator for the export of the small ribosomal subunit to identify necessary genes. Mutations in Ran or its regulators cause 5' ITS1 to accumulate in the nucleoplasm. Accumulation of 5' ITS1 in the nucleus was not found in 28 yeast strains with mutations in other genes that affect traffic in the nucleus.

Uncoupling of the hnRNP Np13p, the main mRNA-binding protein of Saccharomyces cerevisiae, from mRNAs in stress-induced inhibition of mRNA export is discussed. Data that point to an export-incompetent RNP complex without Np13p as the reason for constituitively exported mRNAs' not leaving the nucleus in stressed cells are presented based on seeing that Np13p is exported from the nucleus after onset of strees while mRNA stays nuclear.

An in vivo nuclear export assay has been used to study arginine methylation for nuclear export of the heterogeneous nuclear ribonucleoproteins (hnRNPs). Various hnRNPs help in eukaryotic mRNA processing/export. Both Np13p and Hrp1p fail to leave the nucleus in cells without Hmt1p. Overexpression of Hmt1p helps in Np13p export. A genetic relationship exists between Mnt1p and cap-binding protein 80 (CBP80). Arginine methylation helps in nuclear export of hnRNP proteins. That is at least one of its biological roles. In Saccharomyces cerevisiae, the enzyme mainly responsible for arginine methylation is Hmt1p.

HIV-1 Vpr is similar to importin-B in the function in performs during HIV-1 nuclear import. This function is an essential part of the Vpr role in macrophage infection although not crucial for G2 arrest. The two functions of Vpr are genetically separable. When Vpr does not localize to the nuclear envelope HIV-1 cannot infect macrophages effectively.

Cellular research is continuing to build up an overall picture of nuclear transport mechanisms. The delineating export transportation of molecules from nucleus to cytoplasm are complex compared to nuclear import methods although a greater understanding will inevitably be achieved through further research. Studies into nuclear pores, protein import and nuclear export are described.

Messenger RNA (mRNA) localisation is one of the prime mechanisms to ensure protein localisation in the cytoplasm of polarised embryonic cells, and has been well-studied in the development of Xenopus and Drosophila embryos. But what of other cells? Here, we discuss whether the directed transport of mRNA out of the nucleus, following cytoplasmic highways to a specified organelle in the cytoplasm, might also contribute to the exquisite fidelity of protein targeting observed in all eukaryotic cells.

Research has shown macromolecular movement across the nuclear envelope to be bidirectional, with proteins and RNAs being trafficked through nuclear pore complexes approximately 30 times the size of a ribosome. Of the various processes in nuclear transport, more is known about nuclear protein import than nuclear export. But in the light of several recent discoveries, it is now possible to propose a general mechanism for nuclear transport through the nuclear pore.

Saccharomyces cerevisiae yeast possess a special gene, NPL3, which is responsible for encoding a protein that performs functions similar to heterogeneous nuclear ribonucleoproteins (hnRNP). NPL3 plays an active role in RNA export, import and rRNA processing. Several suppressors contained in the temperature-sensitive allele of NPL3, npl3-1, were isolated and two suppressors were identified with a newly defined gene, HRP1, responsible for encoding a protein similar to hnRNP A/B proteins of vertebrates. It was discovered that mutations in HRP1 suppress all temperature-sensitive npl3 alleles but do not bypass an npl3 null allele.

Yeast Srp1, a protein from Saccharomyces cerevisiae, is needed for protein import and also for the process of mitosis. Cell cycle-regulated proteolysis needs a nuclear import receptor. Initial targeting of nuclear substrate to nuclear envelope in mammalian in vitro assay is done with the help of importin. The sequence similarity and ability of Srp1 to bind to nuclear localization sequence shows that Srp1 and importin are equally important in nuclear import.

Analysis of yeast strains that overexpress a form of Gsp1 with glycine-21 converted to valine (Gsp1-G21V), encoding the GTP-bound form of the Ras-like protein, Ran TC4, shows that the GTP-hydrolyzable and not the GTP-bound form of Ran TC4 is crucial for protein import into the nucleus and the transfer of poly(A)+ RNA from the nucleus to the cytoplasm. Cells containing the GTP-bound form of Ran TC4 have defective nuclear protein localization with the Gsp1-G21V induction by galactose causing accumulation of nuclear proteins in cytoplasm. These cells also retain poly(A)+ RNA in their nuclei.

Five sec63-101 suppressors were isolated to determine components involved in protein trafficking and the role of Sec63p in endoplasmic reticulum translocation and nuclear protein localization. Five complementation groups of mutations were identified and characterized, namely, son1-son5. son1 mutations's suppression of sec63 alleles was found to be characterized by regional specificity. In addition, son1 mutants were found to have slow growth and a partial nuclear antigen mislocalization. Son1 mutations eliminate Son1p function to suppress sec63-101.

Abstract Background FeFe-hydrogenases are the most active class of H2-producing enzymes known in nature and may have important applications in clean H2 energy production. Many potential uses are currently complicated by a crucial weakness: the active sites of all known FeFe-hydrogenases are irreversibly inactivated by O2. Results We have developed a synthetic metabolic pathway in E. coli that links FeFe-hydrogenase activity to the production of the essential amino acid cysteine. Our design includes a complementary host strain whose endogenous redox pool is insulated from the synthetic metabolic pathway. Host viability on a selective medium requires hydrogenase expression, and moderate O2 levels eliminate growth. This pathway forms the basis for a genetic selection for O2 tolerance. Genetically selected hydrogenases did not show improved stability in O2 and in many cases had lost H2 production activity. The isolated mutations cluster significantly on charged surface residues, suggesting the evolution of binding surfaces that may accelerate hydrogenase electron transfer. Conclusions Rational design can optimize a fully heterologous three-component pathway to provide an essential metabolic flux while remaining insulated from the endogenous redox pool. We have developed a number of convenient in vivo assays to aid in the engineering of synthetic H2 metabolism. Our results also indicate a H2-independent redox activity in three different FeFe-hydrogenases, with implications for the future directed evolution of H2-activating catalysts.

Abstract Background The engineering of metabolism holds tremendous promise for the production of desirable metabolites, particularly alternative fuels and other highly reduced molecules. Engineering approaches must redirect the transfer of chemical reducing equivalents, preventing these electrons from being lost to general cellular metabolism. This is especially the case for high energy electrons stored in iron-sulfur clusters within proteins, which are readily transferred when two such clusters are brought in close proximity. Iron sulfur proteins therefore require mechanisms to ensure interaction between proper partners, analogous to many signal transduction proteins. While there has been progress in the isolation of engineered metabolic pathways in recent years, the design of insulated electron metabolism circuits in vivo has not been pursued. Results Here we show that a synthetic hydrogen-producing electron transfer circuit in Escherichia coli can be insulated from existing cellular metabolism via multiple approaches, in many cases improving the function of the pathway. Our circuit is composed of heterologously expressed [Fe-Fe]-hydrogenase, ferredoxin, and pyruvate-ferredoxin oxidoreductase (PFOR), allowing the production of hydrogen gas to be coupled to the breakdown of glucose. We show that this synthetic pathway can be insulated through the deletion of competing reactions, rational engineering of protein interaction surfaces, direct protein fusion of interacting partners, and co-localization of pathway components on heterologous protein scaffolds. Conclusions Through the construction and characterization of a synthetic metabolic circuit in vivo, we demonstrate a novel system that allows for predictable engineering of an insulated electron transfer pathway. The development of this system demonstrates working principles for the optimization of engineered pathways for alternative energy production, as well as for understanding how electron transfer between proteins is controlled.

Encapsulins are recently discovered protein compartments able to specifically encapsulate cargo proteins in vivo. Encapsulation is dependent on C-terminal targeting peptides (TPs). Here, we characterize and engineer TP-shell interactions in the Thermotoga maritima and Myxococcus xanthus encapsulin systems. Using force-field modeling and particle fluorescence measurements we show that TPs vary in native specificity and binding strength, and that TP-shell interactions are determined by hydrophobic and ionic interactions as well as TP flexibility. We design a set of TPs with a variety of predicted binding strengths and experimentally characterize these designs. This yields a set of TPs with novel binding characteristics representing a potentially useful toolbox for future nanoreactor engineering aimed at controlling cargo loading efficiency and the relative stoichiometry of multiple concurrently loaded cargo proteins. [ABSTRACT FROM AUTHOR]