The classical secretion of soluble proteins and transport of integral membrane proteins to the cell surface require transit into and through the endoplasmic reticulum and the Golgi apparatus. Signal peptides or transmembrane domains target proteins for translocation into the lumen or insertion into the membrane of the endoplasmic reticulum, respectively. Here we discuss two mechanisms of unconventional protein targeting to plasma membranes, i.e., transport processes that are active in the absence of a functional Golgi system. We first focus on integral membrane proteins that are inserted into the endoplasmic reticulum but that, however, are transported to plasma membranes in a Golgi-independent manner. We then discuss soluble secretory proteins that are secreted from cells without any involvement of the endoplasmic reticulum and the Golgi apparatus.
Glutathione is central to cellular redox chemistry. The majority of glutathione redox research has been based on the chemical analysis of whole-cell extracts, which unavoidably destroy subcellular compartment-specific information. Compartment-specific real-time measurements based on genetically encoded fluorescent probes now suggest that the cytosolic glutathione redox potential is about 100 mV more reducing than previously thought. Using these probes in yeast, we show that even during severe oxidative stress, the cytosolic glutathione disulfide (GSSG) concentration is much more tightly regulated than expected and provides a mechanistic explanation for the discrepancy with conventional measurements. GSSG that is not immediately reduced in the cytosol is rapidly transported into the vacuole by the ABC-C transporter Ycf1. The amount of whole-cell GSSG is entirely dependent on Ycf1 and uninformative about the cytosolic glutathione pool. Applying these insights, we identify Trx2 and Grx2 as efficient backup systems to glutathione reductase for cytosolic GSSG reduction.
SummaryThe 34 kDa polypeptide of the outer envelope membranes from pea chloroplasts (OEP 34) is a major constituent of this membrane. OEP 34 is detected on polyacrylamide gels under non-reducing condition in association with OEP 75, the putative protein translocation pore. An antiserum against OEP 34 is able to co-immunoprecipitate the precursor of Rubisco small subunit from a partially purified import complex of chloroplast outer envelope membranes. A full-length cDNA clone coding for pea OEP 34 has been isolated. Analysis of the deduced amino acid sequence revealed typical and conserved sequence motifs found in GTP,binding proteins, making it a new and unique member of this superfamily. OEP 34 behaves as an integral constituent of the outer chloroplast envelope, which is anchored by its C-terminus into the membrane, while the majority of the protein projects into the cytoplasm. OEP 34 does not possess a cleavable N-terminal transit sequence but it is targeted to the chloroplasts and integrated into the outer membranes by internal sequence information which seems to be present in the C-terminal membrane anchor region. Productive integration of OEP 34 into the outer envelope requires, in contrast to other OEPs, proteasesensitive chloroplast surface components and is stimulated by ATP. The GTP binding specificity of OEP 34 is demonstrated by photo-affinity labelling in the presence of [~-32p]GTP. Overexpressed and purified OEP 34 possesses endogenous GTPese activity. These results indicate a possible regulatory function of OEP 34 in protein translocation into chloroplasts.
In eukaryotes, termination of messenger RNA (mRNA) translation is mediated by the release factors eRF1 and eRF3. Using Saccharomyces cerevisiae as a model organism, we have identified a member of the DEAD-box protein (DBP) family, the DEAD-box RNA helicase and mRNA export factor Dbp5, as a player in translation termination. Dbp5 interacts genetically with both release factors and the polyadenlyate-binding protein Pab1. A physical interaction was specifically detected with eRF1. Moreover, we show that the helicase activity of Dbp5 is required for efficient stop-codon recognition, and intact Dbp5 is essential for recruitment of eRF3 into termination complexes. Therefore, Dbp5 controls the eRF3-eRF1 interaction and thus eRF3-mediated downstream events.
Scp160p interacts in an mRNA-dependent manner with translating ribosomes via multiple RNA-binding heterogeneous nuclear ribonucleoprotein K-homology (KH) domains. In the present study, we show by protein-protein cross-linking that Scp160p is in close proximity to translation elongation factor 1A and the WD40 (Trp-Asp 40)-repeat containing protein Asc1p at ribosomes. Analysis of a truncation mutant revealed that the C-terminus of Scp160p is essential for ribosome binding and that Cys(1067) at the C-terminus of Scp160p is required to obtain these cross-links. The interaction of Scp160p with ribosomes depends on Asc1p. In fast-growing yeast cells, nearly all Asc1p is tightly bound to ribosomes, but it can also be present in a ribosome-free form depending on growth conditions. The functional homologue of Asc1p, mammalian RACK1 (receptor of activated C kinase), was previously characterized as an adaptor protein bridging activated signalling molecules with their substrates. Our results suggest that Scp160p connects specific mRNAs, ribosomes and a translation factor with an adaptor for signalling molecules. These interactions might regulate the translation activity of ribosomes programmed with specific mRNAs.
The yeast spindle pole body (SPB), the functional equivalent of mammalian centrosome, duplicates in G1/S phase of the cell cycle and then becomes inserted into the nuclear envelope. Here we describe a link between SPB duplication and targeted translation control. When insertion of the newly formed SPB into the nuclear envelope fails, the SESA network comprising the GYF domain protein Smy2, the translation inhibitor Eap1, the mRNAbinding protein Scp160 and the Asc1 protein, specifically inhibits initiation of translation of POM34 mRNA that encodes an integral membrane protein of the nuclear pore complex, while having no impact on other mRNAs. In response to SESA, POM34 mRNA accumulates in the cytoplasm and is not targeted to the ER for cotranslational translocation of the protein. Reduced level of Pom34 is sufficient to restore viability of mutants with defects in SPB duplication. We suggest that the SESA network provides a mechanism by which cells can regulate the translation of specific mRNAs. This regulation is used to coordinate competing events in the nuclear envelope.[Keywords: Regulation of translation; spindle pole body; nuclear pore complex; Smy2; Eap1; Scp160] Supplemental material is available at http://www.genesdev.org.
A major challenge in current molecular biology is to understand how sequential steps in gene expression are coupled. Recently, much attention has been focused on the linkage of transcription, processing, and mRNA export. Here we describe the cytoplasmic rearrangement for shuttling mRNA binding proteins in Saccharomyces cerevisiae during translation. While the bulk of Hrp1p, Nab2p, or Mex67p is not associated with polysome containing mRNAs, significant amounts of the serine/arginine (SR)-type shuttling mRNA binding proteins Npl3p, Gbp2p, and Hrb1p remain associated with the mRNA-protein complex during translation. Interestingly, a prolonged association of Npl3p with polysome containing mRNAs results in translational defects, indicating that Npl3p can function as a negative translational regulator. Consistent with this idea, a mutation in NPL3 that slows down translation suppresses growth defects caused by the presence of translation inhibitors or a mutation in eIF5A. Moreover, using sucrose density gradient analysis, we provide evidence that the import receptor Mtr10p, but not the SR protein kinase Sky1p, is involved in the timely regulated release of Npl3p from polysomeassociated mRNAs. Together, these data shed light onto the transformation of an exporting to a translating mRNP.
The yeast Saccharomyces cerevisiae contains three proteins (Kap104p, Pse1p, and Kap123p) that share similarity to the 95-kDa  subunit of the nuclear transport factor importin (also termed karyopherin and encoded by KAP95͞RSL1 in yeast). Proteins that contain nuclear localization sequences are recognized in the cytoplasm and delivered to the nucleus by the heterodimeric importin complex. A second importin-related protein, transportin, delivers a subset of heterogeneous nuclear ribonucleoproteins (hnRNPs) to the nucleoplasm. We now show that in contrast to loss of importin  (Kap95p͞Rsl1p) and transportin (Kap104p), conditional loss of Pse1p in a strain lacking Kap123p results in a specific block of mRNA export from the nucleus. Overexpression of Sxm1p, a protein related to Cse1p in yeast and to the human cellular apoptosis susceptibility protein, relieves the defects of cells lacking Pse1p and Kap123p. Thus, a major role of Pse1p, Kap123p, and Sxm1p may be nuclear export rather than import, suggesting a symmetrical relationship between these processes.Movement of macromolecules in and out of the nucleus is a highly regulated process essential for proper progression though the cell cycle, response to extracellular signals, and viral maturation. Our knowledge of the mechanism by which proteins are imported into and RNAs exported from the nucleus has grown significantly in recent years. Proteins have been identified that mediate both inward and outward macromolecular movement and both processes appear to be regulated by some of the same factors (reviewed in refs. 1 and 2).Import of proteins into the nucleus is a highly conserved, multistep process initiated by recognition of a nuclear localization sequence (NLS) by a cognate receptor, followed by docking of the complex at the nuclear pore, and GTPdependent translocation into the nucleoplasm. The receptor for the canonical NLS is a heterodimeric protein complex variously called importin or karyopherin, composed of an ␣ (NLS-binding) and a  (docking) subunit (3-6). Translocation through the nuclear pore is driven by GTP hydrolysis, catalyzed by the small ras-related GTP-binding protein Ran and its regulators (7-10). Once inside the nucleus, the importin͞ karyopherin complex is probably dissociated and the transport factors recycled to the cytoplasm (5). This relatively simple model fails to explain the nuclear import of proteins that lack an NLS conforming to the canonical sequences. The recent identification of a role for the importin  homolog transportin, in the nuclear import of heterogeneous nuclear ribonucleoprotein (hnRNP) A1 raised the possibility that different classes of nuclear proteins would be transported by different pathways, defined by the identity of the  subunit involved (11, 12).In contrast to protein import, how RNAs are exported from the nucleus to the cytoplasm is less well understood. Nascent RNA is subject to a number of posttranscriptional modifications, and it seems likely that RNA moves through the nucleoplasm and the nuclear pore...
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