Selective Export Research Articles

Selective Protein Export from the Endoplasmic Reticulum

Selective Protein Export from the Endoplasmic Reticulum

Sec16 Defines Endoplasmic Reticulum Exit Sites and is Required for Secretory Cargo Export in Mammalian Cells

The selective export of proteins and lipids from the endoplasmic reticulum (ER) is mediated by the coat protein complex II (COPII) that assembles onto the ER membrane. In higher eukaryotes, COPII proteins assemble at discrete sites on the membrane known as ER exit sites (ERES). Here, we identify Sec16 as the protein that defines ERES in mammalian cells. Sec16 localizes to ERES independent of Sec23/24 and Sec13/31. Overexpression, and to a lesser extent, small interfering RNA depletion of Sec16, both inhibit ER-to-Golgi transport suggesting that Sec16 is required in stoichiometric amounts. Sar1 activity is required to maintain the localization of Sec16 at discrete locations on the ER membrane, probably through preventing its dissociation. Our data suggest that Sar1-GTP-dependent assembly of Sec16 on the ER membrane forms an organized scaffold defining an ERES.

Selective Export of HLA-F by Its Cytoplasmic Tail

MHC class I molecules exit the endoplasmic reticulum (ER) by an unknown mechanism. Although a selective export mechanism has been proposed for the anterograde transport of class I, a motif responsible for export has never been identified. Although classical class I molecules lacking their cytoplasmic tail are expressed on the cell surface, we found that HLA-F was entirely dependent on its cytoplasmic tail for export from the ER. Two known export motifs were recognizable in HLA-F. A C-terminal valine residue functioned in ER export and interacted with coat complex (COP)II, while an RxR motif also played an important role in anterograde transport and bound to 14-3-3 proteins. This divergent trafficking of HLA-F implicates an alternative function for HLA-F, independent of loading with peptides in the ER.

Adenovirus E1B 55-Kilodalton Protein Is Required for both Regulation of mRNA Export and Efficient Entry into the Late Phase of Infection in Normal Human Fibroblasts

The human adenovirus type 5 (Ad5) E1B 55-kDa protein is required for selective nuclear export of viral late mRNAs from the nucleus and concomitant inhibition of export of cellular mRNAs in HeLa cells and some other human cell lines, but its contributions(s) to replication in normal human cells is not well understood. We have therefore examined the phenotypes exhibited by viruses carrying mutations in the E1B 55-kDa protein coding sequence in normal human fibroblast (HFFs). Ad5 replicated significantly more slowly in HFFs than it does in tumor cells, a difference that is the result of delayed entry into the late phase of infection. The A143 mutation, which specifically impaired export of viral late mRNAs from the nucleus in infected HeLa cells (R. A. Gonzalez and S. J. Flint, J. Virol. 76:4507-4519, 2002), induced a more severe defect in viral mRNA export in HFFs. This observation indicates that the E1B 55-kDa protein regulates mRNA export during the late phase of infection of normal human cells. Other mutants exhibited phenotypes not observed in HeLa cells. In HFFs infected by the null mutant Hr6, synthesis of viral late mRNAs and proteins was severely impaired. Such defects in late gene expression were the result of inefficient progression into the late phase of infection, for viral DNA synthesis was 10-fold less efficient in Hr6-infected HFFs than in cells infected by Ad5. Similar, but less severe, defects in viral DNA synthesis were induced by the insertion mutation H224, which has been reported to inhibit binding of the E1B 55-kDa protein to p53 (C. C. Kao, P. R. Yew, and A. J. Berk, Virology 179:806-814, 1990).

Selective Golgi export of Kir2.1 controls the stoichiometry of functional Kir2.x channel heteromers

Surface expression of ion channels and receptors often depends on intrinsic sequence motifs that control their intracellular transport along the secretory pathway. Although members of the Kir2.x subfamily share two such motifs - a diacidic ER export motif and a positively charged Golgi export motif - they strongly differ in their surface expression. Whereas Kir2.1 shows prominent plasma membrane localization, Kir2.4 channels accumulate within the Golgi complex. By constructing chimeras between Kir2.1 and Kir2.4 subunits, a stretch of 20 amino acids was identified in the Kir2.1 C-terminus that is both necessary and sufficient to promote anterograde transport of Kir channel subunits at the level of trafficking from the Golgi to the plasma membrane. The core element of the identified sequence bears a tyrosine-dependent YXXPhi consensus motif for adaptin binding, with the flanking residues determining its functional efficiency. As the signal is dominant in promoting surface transport of Kir2.1/Kir2.4 channel heteromers and is recognized by both the epithelial and neuronal intracellular sorting machinery, the preferential Golgi export of Kir2.1 will control the stoichiometry of Kir2.x heteromers expressed on the cell surface.

COPII Coat Assembly and Selective Export from the Endoplasmic Reticulum

The coat protein complex II (COPII) generates transport vesicles that mediate protein transport from the endoplasmic reticulum (ER). Recent structural and biochemical studies have suggested that the COPII coat is responsible for direct capture of membrane cargo proteins and for the physical deformation of the ER membrane that drives the transport vesicle formation. The COPII-coated vesicle formation at the ER membrane is triggered by the activation of the Ras-like small GTPase Sar1 by GDP/GTP exchange, and activated Sar1 in turn promotes COPII coat assembly. Subsequent GTP hydrolysis by Sar1 leads to disassembly of the coat proteins, which are then recycled for additional rounds of vesicle formation. Thus, the Sar1 GTPase cycle is thought to regulate COPII coat assembly and disassembly. Emerging evidence suggests that the cargo proteins modulate the Sar1 GTP hydrolysis to coordinate coat assembly with cargo selection. Here, I discuss the possible roles of the GTP hydrolysis by Sar1 in COPII coat assembly and selective uptake of cargo proteins into transport vesicles.

Nucleolar localization of human hepatitis B virus capsid protein.

Wild-type human hepatitis B virus (HBV) exhibits selective export of virions containing mature genomes. In contrast, changing an isoleucine to a leucine at amino acid 97 (I97L) of the HBV core antigen (HBcAg) causes it to release immature genomes. To elucidate the structure-function relationship of HBcAg at amino acid 97, we systematically replaced the isoleucine residue at this position with 18 other amino acids via mutagenesis. Twelve of the 18 mutants exhibited no significant phenotype, while five new mutants displayed strong phenotypes. The I97D mutant had a near lethal phenotype, the I97P mutant exhibited a significantly reduced level of virion secretion, and the I97G mutant lacked the full-length relaxed circular form of viral DNA. The tip of the spike of the capsid particle is known to contain a predominant B-cell epitope. However, the recognition of this exposed epitope by an anti-HBc antibody appeared to be affected by the I97E mutation or by histidine tagging at the C terminus of mutant HBcAg, which is presumably in the capsid interior. Surprisingly, the nuclear HBcAg of mutants I97E and I97W, produced from either a replicon or an expression vector, was found to be colocalized with nucleolin and B23 at a frequency of nearly 100% by confocal immunofluorescence microscopy. In contrast, this colocalization occurred with wild-type HBcAg only to a limited extent. We also noted that nucleolin-colocalizing cells were often binucleated or apoptotic, suggesting that the presence of HBcAg in the nucleolus may perturb cytokinesis. The mechanism of this phenomenon and its potential involvement in liver pathogenesis are discussed. To our knowledge, this is the first report of nucleolar HBcAg in culture.

Stress response in yeast mRNA export factor: reversible changes in Rat8p localization are caused by ethanol stress but not heat shock.

Ethanol stress (10% v/v) causes selective mRNA export in Saccharomyces cerevisiae in a similar manner to heat shock (42 degrees C). Bulk poly(A)(+) mRNA accumulates in the nucleus, whereas heat shock protein mRNA is exported under such conditions. Here we investigated the effects of stress on mRNA export factors. In cells treated with ethanol stress, the DEAD box protein Rat8p showed a rapid and reversible change in its localization, accumulating in the nucleus. This change correlated closely with the blocking of bulk poly(A)(+) mRNA export caused by ethanol stress. We also found that the nuclear accumulation of Rat8p is caused by a defect in the Xpo1p/Crm1p exportin. Intriguingly, the localization of Rat8p did not change in heat shocked cells, suggesting that the mechanisms blocking bulk poly(A)(+) mRNA export differ for heat shock and ethanol stress. These results suggest that changes in the localization of Rat8p contribute to the selective export of mRNA in ethanol stressed cells, and also indicate differences in mRNA export between the heat shock response and ethanol stress response.

Effect of placental function on fatty acid requirements during pregnancy.

The fetus has an absolute requirement for the n-3/n-6 fatty acids and docosahexaenoic acid (22:6 n-3; DHA) in particular is essential for the development of the brain and retina. Most of the fat deposition in the fetus occurs in the last 10 weeks of pregnancy. The likely rate of DHA utilisation during late pregnancy cannot be met from dietary sources alone in a significant proportion of mothers. De novo synthesis makes up some of the shortfall but the available evidence suggests that the maternal adipose tissue makes a significant contribution to placental transport to the fetus. The placenta plays a crucial role in mobilising the maternal adipose tissue and actively concentrating and channelling the important n-3/n-6 fatty acids to the fetus via multiple mechanisms including selective uptake by the syncytiotrophoblast, intracellular metabolic channelling, and selective export to the fetal circulation. These mechanisms protect the fetus against low long-chain polyunsaturated fatty acid (LCPUFA) intakes in the last trimester of pregnancy and have the effect of reducing the maternal dietary requirement for preformed DHA at this time. As a result of these adaptations, small changes in the composition of the habitual maternal diet before pregnancy are likely to be more effective in improving LCPUFA delivery to the fetus than large dietary changes in late pregnancy. There is little evidence that DHA intake/status in the second half of pregnancy affects visual and cognitive function in the offspring, but more studies are needed, particularly in children born to vegetarian and vegan and mothers who may have very low intakes of DHA.

FXYD7, Mapping of Functional Sites Involved in Endoplasmic Reticulum Export, Association With and Regulation of Na,K-ATPase

The brain-specific FXYD7 is a member of the recently defined FXYD family that associates with the alpha1-beta1 Na,K-ATPase isozyme and induces an about 2-fold decrease in its apparent K+ affinity. By using the Xenopus oocyte as an expression system, we have investigated the role of conserved and FXYD7-specific amino acids in the cellular routing of FXYD7 and in its association with and regulation of Na,K-ATPase. In contrast to FXYD2 and FXYD4, the studies on FXYD7 show that the conserved FXYD motif in the extracytoplasmic domain is not involved in the efficient association of FXYD7 with Na,K-ATPase. On the other hand, the conserved Gly40 and Gly29, located on the same face of the transmembrane helix, were found to be implicated both in the association with and the regulation of Na,K-ATPase. Mutational analysis of FXYD7-specific regions revealed the presence of an ER export signal at the end of the cytoplasmic tail. Deletion of a C-terminal valine residue in FXYD7 significantly delayed and decreased its O-glycosylation processing and retarded the rate of its cell surface expression. This result indicates that the C-terminal valine residue is involved in the rapid and selective ER export of FXYD7, which could explain the observed post-translational association of FXYD7 with Na,K-ATPase. In conclusion, our study on FXYD7 provides new information on structural determinants of general importance for FXYD protein action. Moreover, FXYD7 is identified as a new member of proteins with a regulated ER export, which suggests that, among FXYD proteins, FXYD7 has a particular regulatory function in brain.

Protein Profiling with Cleavable Isotope-coded Affinity Tag (cICAT) Reagents: The Yeast Salinity Stress Response

Protein expression profiles in yeast cells, in response to salinity stress, were determined using the cleavable isotope-coded affinity tag (cICAT) labeling strategy. The analysis included separation of the mixed protein samples by SDS-PAGE, followed by excision of the entire gel lane, and division of the lane into 14 gel regions. Regions were subjected to in-gel digestion, biotin affinity chromatography, and analysis by nano-scale microcapillary liquid chromatography coupled to tandem mass spectrometry. The novel (13)C-labeled ICAT reagents have identical elution profiles for labeled peptide pairs and broadly spread the distribution of labeled peptides during reversed-phase chromatography. A total of 560 proteins were identified and quantified, with 51 displaying more than 2-fold expression differences. In addition to some known proteins involved in salt stress, four RNA-binding proteins were found to be up-regulated by high salinity, suggesting that selective RNA export from the nucleus is important for the salt-stress response. Some proteins involved in amino acid synthesis, which have been observed to be up-regulated by amino acid starvation, were also found to increase their abundance on salt stress. These results indicate that salt stress and amino acid starvation cause overlapping cellular responses and are likely to be physiologically linked.

ER export of ERGIC-53 is controlled by cooperation of targeting determinants in all three of its domains.

Selective export of proteins from the endoplasmic reticulum (ER) requires transport signals that have not been fully characterized. Here, we provide the first complete map of ER export determinants of a type I membrane protein, ERGIC-53, that cycles in the early secretory pathway. ER export requires a phenylalanine motif at the C-terminus, known to mediate coat protein II (COPII) interaction, that is assisted by a glutamine in the cytoplasmic domain. Disulfide bond-stabilized oligomerization is also required. Efficient hexamerization depends on the presence of a polar and two aromatic residues in the transmembrane domain (TMD). Oligomerization becomes independent on disulfide bonds when TMD hydrophobicity is increased. ER export is also influenced by TMD length, 21 amino acids being most efficient. When transferred to a signal-less construct, the established targeting motifs reconstitute full transport activity. The results suggest an ER-export mechanism in which transmembrane and luminal determinants mediate oligomerization required for efficient recruitment of ERGIC-53 into budding vesicles via the C-terminal COPII-binding phenylalanine motif.

Surface Expression of Inward Rectifier Potassium Channels Is Controlled by Selective Golgi Export

Traffic of integral membrane proteins along the secretory pathway is not simply a default process but can be selective. Such selectivity is achieved by sequence information within the cargo protein that recruits coat protein complexes to drive the formation of transport vesicles. A number of sequence motifs have been identified in the cytoplasmic domains of ion channels that regulate early trafficking events between the endoplasmic reticulum and the Golgi complex. Here, we demonstrate that the following trafficking step from the Golgi compartment to the plasma membrane can also be selective. The N-terminal domain of the inward rectifier potassium channel Kir2.1 contains specific sequence information that is necessary for its efficient export from the Golgi complex. Lack of this information results in accumulation of the protein within the Golgi and a significant decrease in cell surface expression. As similar results were obtained for the N terminus of another Kir channel subfamily member, Kir4.1, which could functionally substitute for the Kir2.1 N terminus, we propose a more general role of the identified N-terminal domains for post-Golgi trafficking of Kir channels.

Regulation of mRNA production by the adenoviral E1B 55-kDa and E4 Orf6 proteins.

The E1B 55-kDa and E4 Orf6 proteins of human subgroup C adenoviruses both counter host cell defenses mediated by the cellular p53 protein and regulate viral late gene expression. A complex containing the two proteins has been implicated in induction of selective export of viral late mRNAs from the nucleus to the cytoplasm, with concomitant inhibition of export of the majority of newly synthesized cellular mRNAs. The molecular mechanisms by which these viral proteins subvert cellular pathways of nuclear export are not yet clear. Here, we review recent efforts to identify molecular and biochemical functions of the E1B 55-kDa and E4 Orf6 proteins required for regulation of mRNA export, the several difficulties and discrepancies that have been encountered in studies of these viral proteins, and evidence indicating that the reorganization of the infected cell nucleus and production of viral late mRNA at specific intra-nuclear sites are important determinants of selective mRNA export in infected cells. In our view, it is not yet possible to propose a coherent molecular model for regulation of mRNA export by the E1B 55-kDa and E4 Orf6 proteins. However, it should now be possible to address specific questions about the roles of potentially relevant properties of these viral proteins.

ER-to-Golgi transport: COP I and COP II function (Review).

COP I and COP II coat proteins direct protein and membrane trafficking in between early compartments of the secretory pathway in eukaryotic cells. These coat proteins perform the dual, essential tasks of selecting appropriate cargo proteins and deforming the lipid bilayer of appropriate donor membranes into buds and vesicles. COP II proteins are required for selective export of newly synthesized proteins from the endoplasmic reticulum (ER). COP I proteins mediate a retrograde transport pathway that selectively recycles proteins from the cis-Golgi complex to the ER. Additionally, COP I coat proteins have complex functions in intra-Golgi trafficking and in maintaining the normal structure of the mammalian interphase Golgi complex.

Influence of a putative intermolecular interaction between core and the pre-S1 domain of the large envelope protein on hepatitis B virus secretion.

Virion release of hepatitis B virus (HBV) from hepatocytes is a tightly regulated event. It is a dogma that only the mature HBV genome is preferentially allowed to export from the intracellular compartment (J. Summers and W. S. Mason, Cell 29:403-415, 1982). Recently, an "immature secretion" phenotype of a highly frequent naturally occurring HBV variant containing a leucine residue at amino acid 97 of the core protein was identified. Unlike wild-type HBV, this variant secretes almost equal amounts of mature and immature genomes. This phenomenon is not caused by any instability of core particles or by any deficiency in viral reverse transcription (T. T. Yuan, P. C. Tai, and C. Shih, J. Virol. 73:10122-10128, 1999). In this study, our kinetic analysis of virion secretion of the mutant F97L (phenylalanine to leucine) indicates that the secretion of its immature genome does not occur earlier than that of its mature genome. In addition, the secretion kinetics of the mature genomes are comparable between the wild-type HBV and the mutant F97L. Therefore, the immature secretion phenomenon of mutant F97L is not caused by premature secretion or more efficient secretion. Previously, we hypothesized that the immature secretion phenotype is probably caused by the aberrant interaction between its mutant core and wild-type envelope proteins. Here, we further demonstrated that a pre-S1 envelope mutation at position 119, changing an alanine (A) to a phenylalanine (F), can offset the immature secretion phenotype of the mutant I97L (isoleucine to leucine) and successfully restore the wild-type-like selective export of the mature genome of the double mutant pre-S1-A119F/core-I97L.

Effects of mutations in the adenoviral E1B 55-kilodalton protein coding sequence on viral late mRNA metabolism.

The human subgroup C adenoviral E1B 55-kDa protein cooperates with the viral E4 Orf6 protein to induce selective export of viral, late mRNAs from the nucleus to the cytoplasm. Previous studies have suggested that such preferential transport of viral mRNA and the concomitant inhibition of export of cellular mRNAs are the result of viral colonization of specialized microenvironments within the nucleus. However, neither the molecular basis of this phenomenon nor the mechanism by which the E1B 55-kDa protein acts has been elucidated. We therefore examined viral late mRNA metabolism in HeLa cells infected with a series of mutant viruses that carry insertions at various positions in the E1B protein coding sequence (P. R. Yew, C. C. Kao, and A. J. Berk, Virology 179:795-805, 1990). All the mutations examined impaired cytoplasmic accumulation of viral L2 mRNAs and reduced L2 mRNA export efficiency. However, in most cases these defects could be ascribed to reduced E1B 55-kDa protein concentration or the unexpected failure of the altered E1B proteins to enter the nucleus efficiently. The latter property, the pleiotropic defects associated with all the mutations that impaired nuclear entry of the E1B protein, and consideration of its primary sequence suggest that these insertions result in misfolding of the protein. Insertion of four amino acids at residue 143 also inhibited viral mRNA export but resulted in increased rather than decreased accumulation of the E1B 55-kDa protein in the nucleus. This mutation specifically impaired the previously described association of the E1B protein with intranuclear structures that correspond to sites of adenoviral DNA replication and transcription (D. Ornelles and T. Shenk, J. Virol. 65:424-439, 1991) and the colocalization of the E1B and E4 Orf6 proteins. As this insertion has been shown to inhibit the interaction of the E1B with the E4 Orf6 protein in infected cell extracts (S. Rubenwolf, H. Schütt, M. Nevels, H. Wolf, and T. Dobner, J. Virol. 71:1115-1123, 1997), these phenotypes provide direct support for the hypothesis that selective viral mRNA export is determined by the functional organization of the infected cell nucleus.

Genomic but not antigenomic hepatitis delta virus RNA is preferentially exported from the nucleus immediately after synthesis and processing.

Hepatitis delta virus (HDV) contains a viroid-like circular RNA that replicates via a double rolling circle replication mechanism. It is generally assumed that HDV RNA is synthesized and remains exclusively in the nucleus until being exported to the cytoplasm for virion assembly. Using a [32P]orthophosphate metabolic labeling procedure to study HDV RNA replication (T. B. Macnaughton, S. T. Shi, L. E. Modahl, and M. M. C. Lai. J. Virol. 76:3920-3927, 2002), we unexpectedly found that a significant amount of newly synthesized HDV RNA was detected in the cytoplasm. Surprisingly, Northern blot analysis revealed that the genomic-sense HDV RNA is present almost equally in both the nucleus and cytoplasm, whereas antigenomic HDV RNA was mostly retained in the nucleus, suggesting the specific and highly selective export of genomic HDV RNA. Kinetic studies showed that genomic HDV RNA was exported soon after synthesis. However, only the monomer and, to a lesser extent, the dimer HDV RNAs were exported to the cytoplasm; very little higher-molecular-weight HDV RNA species were detected in the cytoplasm. These results suggest that the cleavage and processing of HDV RNA may facilitate RNA export. The export of genomic HDV RNA was resistant to leptomycin B, indicating that a cell region maintenance 1 (Crm1)-independent pathway was involved. The large form of hepatitis delta antigen (L-HDAg), which is responsible for virus packaging, was not required for RNA export, as a mutant HDV RNA genome unable to synthesize L-HDAg was still exported. The proportions of genomic HDV RNA in the nucleus and cytoplasm remained relatively constant throughout replication, indicating that export of genomic HDV RNA occurred continuously. In contrast, while antigenomic HDV RNA was predominantly in the nucleus, there was a proportionally large fraction of antigenomic HDV RNA in the cytoplasm at early time points of RNA replication. These findings uncover a previously unrecognized presence of HDV RNA in the cytoplasm, which may have implications for viral RNA synthesis and packaging.

Placental Regulation of Fatty Acid Delivery and its Effect on Fetal Growth—A Review

More than 90 per cent of the fat deposition in the fetus occurs in the last 10 weeks of pregnancy during which it increases exponentially to reach a rate of accretion of around 7g/day close to term. All of the n -3 andn -6 fatty acid structure acquired by the fetus has to cross the placenta and fetal blood is enriched in long chain polyunsaturated fatty acids (LCPUFA) relative to the maternal supply. The placenta may regulate its own fatty acid substrate supply via the action of placental leptin on maternal adipose tissue. Fatty acids cross the microvillous and basal membranes by simple diffusion and via the action of membrane bound and cytosolic fatty acid binding proteins (FABPs). The direction and magnitude of fatty acid flux is mainly dictated by the relative abundance of available binding sites. The fatty acid mix delivered to the fetus is largely determined by the fatty acid composition of the maternal blood although the placenta is able to preferentially transfer the important PUFA to the fetus as a result of selective uptake by the syncytiotrophoblast, intracellular metabolic channelling of individual fatty acids, and selective export to the fetal circulation. Placental FABP polymorphisms may affect these processes. There is little evidence to suggest that placental delivery of fatty acids limits normal fetal growth although the importance of the in utero supply may be to support post-natal development as most of the LCPUFA accumulated by the fetus is stored in the adipose tissue for use in early post-natal life.

Communication of the position of exon-exon junctions to the mRNA surveillance machinery by the protein RNPS1.

In mammalian cells, splice junctions play a dual role in mRNA quality control: They mediate selective nuclear export of mature mRNA and they serve as a mark for mRNA surveillance, which subjects aberrant mRNAs with premature termination codons to nonsense-mediated decay (NMD). Here, we demonstrate that the protein RNPS1, a component of the postsplicing complex that is deposited 5' to exon-exon junctions, interacts with the evolutionarily conserved human Upf complex, a central component of NMD. Significantly, RNPS1 triggers NMD when tethered to the 3' untranslated region of beta-globin mRNA, demonstrating its role as a subunit of the postsplicing complex directly involved in mRNA surveillance.