A novel direct interaction of endoplasmic reticulum with microtubules

The positioning and dynamics of organelles in eukaryotic cells critically depend on membrane-cytoskeleton interactions. Motor proteins play an important role in the directed movement of organelle membranes along microtubules, but the basic mechanism by which membranes stably interact with the microtubule cytoskeleton is largely unknown. Here we report that p63, an integral membrane protein of the reticular subdomain of the rough endoplasmic reticulum (ER), binds microtubules in vivo and in vitro. Overexpression of p63 in cell culture led to a striking rearrangement of the ER and to concomitant bundling of microtubules along the altered ER. Mutational analysis of the cytoplasmic domain of p63 revealed two determinants responsible for these changes: an ER rearrangement determinant near the N-terminus and a central microtubule-binding region. The two determinants function independently of one another as indicated by deletion experiments. A peptide corresponding to the cytoplasmic tail of p63 promoted microtubule polymerization in vitro. p63 is the first identified integral membrane protein that can link a membrane organelle directly to microtubules. By doing so, it may contribute to the positioning of the ER along microtubules.     

Kinetics of fusion between endoplasmic reticulum vesicles in vitro

The endoplasmic reticulum (ER) is a highly dynamic organelle, continuously undergoing membrane fusion and fission. We have measured homotypic fusion between ER vesicles isolated from Chinese hamster ovary cells kinetically in vitro, using an assay based on the metabolic incorporation of pyrene-labeled fatty acids into the phospholipids of cellular membranes. An increase in pyrene-monomer fluorescence was observed after mixing labeled and unlabeled ER vesicles in the presence of ATP and GTP. The protein, temperature, and nucleotide dependence of the increase indicated that it was caused by membrane fusion rather than molecular transfer of labeled lipids to unlabeled membranes. This assay allowed the first kinetic measurements with virtually nonexchangeable probes of a homotypic membrane fusion event. At 37 degrees C, fusion started off immediately at a rate of 1.14 +/- 0.29%/min and reached a half-maximal level after 56 min. In the presence of guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS), or after treatment of the membranes with N-ethylmaleimide, fusion was reduced but not completely inhibited. Addition of GTP during a fusion reaction immediately accelerated, and GTPgammaS immediately slowed down the fusion reaction. Thus, these kinetic measurements indicate that G-proteins might act to rapidly enhance fusion beyond a basic level.     

Similar processes mediate glycopeptide export from the endoplasmic reticulum in mammalian cells and Saccharomyces cerevisiae

Glycopeptides are transported from the lumen of the yeast endoplasmic reticulum (ER) to the cytosol and in contrast to secretory proteins do not enter ER-to-Golgi transport vesicles. In a cell-free system, this process is ATP- and cytosol-dependent. While yeast cytosol promotes the export of glycopeptides from mammalian ER in vitro, glycopeptide release cannot be detected in the presence of mammalian cytosol. We demonstrate that this is due to an N-glycanase activity in mammalian cytosol rather than lack of glycopeptide transport activity in mammalian microsomes. Monitoring the amount of glycopeptide enclosed in ER membranes we show the cytosol- and ATP-dependent release of glycopeptide from mammalian microsomes. The fact that glycopeptide export can be achieved with ER and cytosol derived from heterologous sources indicates that glycopeptide export from the ER is an important process conserved during evolution.     

Topology of Euglena chloroplast protein precursors within endoplasmic reticulum to Golgi to chloroplast transport vesicles

Euglena chloroplast protein precursors are transported as integral membrane proteins from the endoplasmic reticulum (ER) to the Golgi apparatus prior to chloroplast localization. All Euglena chloroplast protein precursors have functionally similar bipartite presequences composed of an N-terminal signal peptide domain and a stromal targeting domain containing a hydrophobic region approximately 60 amino acids from the predicted signal peptidase cleavage site. Asparagine-linked glycosylation reporters and presequence deletion constructs of the precursor to the Euglena light-harvesting chlorophyll a/b-binding protein of photosystem II (pLHCPII) were used to identify presequence regions translocated into the ER lumen and stop transfer membrane anchor domains. An asparagine-linked glycosylation site present at amino acid 148 of pLHCPII near the N terminus of mature LHCPII was not glycosylated in vitro by canine microsomes while an asparagine-linked glycosylation site inserted at amino acid 40 was. The asparagine at amino acid 148 was glycosylated upon deletion of amino acids 46-146, which contain the stromal targeting domain, indicating that the hydrophobic region within this domain functions as a stop transfer membrane anchor sequence. Protease protection assays indicated that for all constructs, mature LHCPII was not translocated across the microsomal membrane. Taken together with the structural similarity of all Euglena presequences, these results demonstrate that chloroplast precursors are anchored within ER and Golgi transport vesicles by the stromal targeting domain hydrophobic region oriented with the presequence N terminus formed by signal peptidase cleavage in the vesicle lumen and the mature protein in the cytoplasm.     

Bcl-XL cooperatively associates with the Bap31 complex in the endoplasmic reticulum, dependent on procaspase-8 and Ced-4 adaptor

Bap31 is a polytopic integral membrane protein of the endoplasmic reticulum and forms a complex with Bcl-2/Bcl-XL and procaspase-8 (Ng, F. W. H., Nguyen, M., Kwan, T., Branton, P. E., Nicholson, W. D., Cromlish, J. A., and Shore, G. C. (1997) J. Cell Biol. 139, 327-338). In co-transfected human cells, procaspase-8 is capable of interacting with Ced-4, an important adaptor molecule in Caenorhabditis elegans that binds to and activates the C. elegans procaspase, proCed-3. Here, we show that the predicted death effector homology domain within the cytosolic region of Bap31 interacts with Ced-4 and contributes to recruitment of procaspase-8. Bcl-XL, which binds directly but weakly to the polytopic transmembrane region of Bap31, indirectly and cooperatively associates with the Bap31 cytosolic domain, dependent on the presence of procaspase-8 and Ced-4. Ced-4Deltac does not interact with Bcl-XL but rather displaces it from Bap31, suggesting that an endogenous Ced-4-like adaptor is a normal constituent of the Bap31 complex and is required for stable association of Bcl-XL with Bap31 in vivo. These findings indicate that Bap31 is capable of recruiting essential components of a core death regulatory machinery.     

The endoplasmic reticulum of plant cells and its role in protein maturation and biogenesis of oil bodies

The endoplasmic reticulum (ER) is the port of entry of proteins into the endomembrane system, and it is also involved in lipid biosynthesis and storage. This organelle contains a number of soluble and membrane-associated enzymes and molecular chaperones, which assist the folding and maturation of proteins and the deposition of lipid storage compounds. The regulation of translocation of proteins into the ER and their subsequent maturation within the organelle have been studied in detail in mammalian and yeast cells, and more recently also in plants. These studies showed that in general the functions of the ER in protein synthesis and maturation have been highly conserved between the different organisms. Yet, the ER of plants possesses some additional functions not found in mammalian and yeast cells. This compartment is involved in cell to cell communication via the plasmodesmata, and, in specialized cells, it serves as a storage site for proteins. The plant ER is also equipped with enzymes and structural proteins which are involved in the process of oil body biogenesis and lipid storage. In this review we discuss the components of the plant ER and their function in protein maturation and biogenesis of oil bodies. Due to the large number of cited papers, we were not able to cite all individual references and in many cases we refer the readers to reviews and references therein. We apologize to the authors whose references are not cited.     

Association of spectrin with a subcompartment of the endoplasmic reticulum in honeybee photoreceptor cells

In order to investigate the mechanisms involved in originating a diverse TCR repertoire in human peripheral blood we analyzed TCRV beta surface expression in different T cell subsets of unrelated individuals. The relative frequencies of 11 distinct V beta chains were determined for immature double positive (DP) as well as for mature CD4 single positive (4SP) and CD8 single positive (8SP) thymocytes, respectively. By comparing these data with expression in peripheral blood T lymphocytes of the same donors we were able to show that usage of TCRV beta in peripheral T cells is significantly (p < 0.001) depending on the pattern in mature SP thymocytes whereas the frequency of TCRV beta families in immature DP thymocytes has no impact (p > 0.2). No association with distinct HLA-haplotypes was observed. Preferential usage of V beta-families in either CD4- or CD8-positive peripheral T cells also correlates with the status in mature thymic precursors (p < 0.001). Altogether, this first combined study of TCR frequencies within different stages of human T cell ontogeny indicates that TCRV beta repertoire is determined mainly through selectional processes within the thymus. Since neither genomically imposed expression nor modulating events in the periphery seem to have strong influence on the relative expression of TCRV beta chains these findings have to be considered in future studies of human diseases.     

Pituitary tumor-transforming gene protein associates with ribosomal protein S10 and a novel human homologue of DnaJ in testicular cells

Pituitary tumor-transforming gene (PTTG) is a recently characterized proto-oncogene that is expressed specifically in adult testis. In this study, we have used in situ hybridization and developmental Northern blot assays to demonstrate that PTTG mRNA is expressed stage-specifically in spermatocytes and spermatids during rat spermatogenic cycle. We have used the yeast two-hybrid system to identify proteins that interact with PTTG in testicular cells. Two positive clones were characterized. One of the clones is the ribosomal protein S10, the other encodes a novel human DnaJ homologue designated HSJ2. Northern blot analysis showed that testis contains higher levels of HSJ2 mRNA than other tissues examined, and the expression pattern of HSJ2 mRNA in postnatal rat testis is similar to PTTG. S10 mRNA levels do not vary remarkably among different tissues and remains unchanged during testicular germ cell differentiation. In vitro binding assays demonstrated that both S10 and HSJ2 bind to PTTG specifically and that PTTG can be co-immunoprecipitated with S10 and HSJ2 from transfected cells. Moreover, the binding sites for both proteins were located within the C-terminal 75 amino acids of the PTTG protein. These results suggest that PTTG may play a role in spermatogenesis.     

Insertion of a bacterial secondary transport protein in the endoplasmic reticulum membrane

The sodium ion-dependent citrate carrier of Klebsiella pneumoniae (CitS) contains 12 hydrophobic potential transmembrane domains. Surprisingly, an alkaline phosphatase fusion study in Escherichia coli has suggested that only 9 of these domains are embedded in the membrane, and 3 are translocated to the periplasm (van Geest, M., and Lolkema, J. S. (1996) J. Biol. Chem. 271, 25582-25589). To provide independent data on the topology and mode of membrane insertion of CitS, we have investigated its insertion into the endoplasmic reticulum (ER) membrane. By using in vitro translation of model proteins in the presence of dog pancreas microsomes, each of the putative transmembrane segments of CitS was assayed for its potency to insert into the ER membrane, both as an isolated segment as well as in the context of COOH-terminal truncation mutants. All 12 segments were able to insert into the membrane as Ncyt-Clum signal anchor sequences. In a series of COOH-terminal truncation mutants, the segments inserted in a sequential way except for one segment, segment Vb, which was translocated to the lumen. Hydrophobic segments VIII and IX, which, according to the alkaline phosphatase fusion study, are in the periplasm of E. coli, form a helical hairpin in the ER membrane. These observations suggest a topology for CitS with 11 transmembrane segments and also demonstrate that the sequence requirements for signal anchor and stop transfer function are different.     

A novel form of hereditary myeloperoxidase deficiency linked to endoplasmic reticulum/proteasome degradation

Myeloperoxidase (MPO) deficiency is a common inherited disorder linked to increased susceptibility to infection and malignancy. We identified a novel missense mutation in the MPO gene at codon 173 whereby tyrosine is replaced with cysteine (Y173C) that is associated with MPO deficiency and assessed its impact on MPO processing and targeting in transfectants expressing normal or mutant proteins. Although the precursor synthesized by cells expressing the Y173C mutation (MPOY173C) was glycosylated, associated with the molecular chaperones calreticulin and calnexin, and acquired heme, it was neither proteolytically processed to mature MPO subunits nor secreted. After prolonged association with calreticulin and calnexin in the endoplasmic reticulum, MPOY173C was degraded. Furthermore, the 20S proteasome inhibitor N-acetyl-L-leucinyl-L-leucinyl-L-norleucinyl inhibited its degradation, suggesting that the proteasome mediates proteolysis of MPOY173C and, thus, participates in quality control in this novel form of hereditary MPO deficiency.     

A role for the DnaJ homologue Scj1p in protein folding in the yeast endoplasmic reticulum

Members of the eukaryotic heat shock protein 70 family (Hsp70s) are regulated by protein cofactors that contain domains homologous to bacterial DnaJ. Of the three DnaJ homologues in the yeast rough endoplasmic reticulum (RER; Scj1p, Sec63p, and Jem1p), Scj1p is most closely related to DnaJ, hence it is a probable cofactor for Kar2p, the major Hsp70 in the yeast RER. However, the physiological role of Scj1p has remained obscure due to the lack of an obvious defect in Kar2p-mediated pathways in scj1 null mutants. Here, we show that the Deltascj1 mutant is hypersensitive to tunicamycin or mutations that reduce N-linked glycosylation of proteins. Although maturation of glycosylated carboxypeptidase Y occurs with wild-type kinetics in Deltascj1 cells, the transport rate for an unglycosylated mutant carboxypeptidase Y (CPY) is markedly reduced. Loss of Scj1p induces the unfolded protein response pathway, and results in a cell wall defect when combined with an oligosaccharyltransferase mutation. The combined loss of both Scj1p and Jem1p exaggerates the sensitivity to hypoglycosylation stress, leads to further induction of the unfolded protein response pathway, and drastically delays maturation of an unglycosylated reporter protein in the RER. We propose that the major role for Scj1p is to cooperate with Kar2p to mediate maturation of proteins in the RER lumen.     

Localization of Xenopus Vg1 mRNA by Vera protein and the endoplasmic reticulum

In many organisms, pattern formation in the embryo develops from the polarized distributions of messenger RNAs (mRNAs) in the egg. In Xenopus, the mRNA encoding Vg1, a growth factor involved in mesoderm induction, is localized to the vegetal cortex of oocytes. A protein named Vera was shown to be involved in Vg1 mRNA localization. Vera cofractionates with endoplasmic reticulum (ER) membranes, and endogenous Vg1 mRNA is associated with a subcompartment of the ER. Vera may promote mRNA localization in Xenopus oocytes by mediating an interaction between the Vg1 3' untranslated region and the ER subcompartment.     

The Golgi and endoplasmic reticulum remain independent during mitosis in HeLa cells

Partitioning of the mammalian Golgi apparatus during cell division involves disassembly at M-phase. Despite the importance of the disassembly/reassembly pathway in Golgi biogenesis, it remains unclear whether mitotic Golgi breakdown in vivo proceeds by direct vesiculation or involves fusion with the endoplasmic reticulum (ER). To test whether mitotic Golgi is fused with the ER, we compared the distribution of ER and Golgi proteins in interphase and mitotic HeLa cells by immunofluorescence microscopy, velocity gradient fractionation, and density gradient fractionation. While mitotic ER appeared to be a fine reticulum excluded from the region containing the spindle-pole body, mitotic Golgi appeared to be dispersed small vesicles that penetrated the area containing spindle microtubules. After cell disruption, M-phase Golgi was recovered in two size classes. The major breakdown product, accounting for at least 75% of the Golgi, was a population of 60-nm vesicles that were completely separated from the ER using velocity gradient separation. The minor breakdown product was a larger, more heterogenously sized, membrane population. Double-label fluorescence analysis of these membranes indicated that this portion of mitotic Golgi also lacked detectable ER marker proteins. Therefore we conclude that the ER and Golgi remain distinct at M-phase in HeLa cells. To test whether the 60-nm vesicles might form from the ER at M-phase as the result of a two-step vesiculation pathway involving ER-Golgi fusion followed by Golgi vesicle budding, mitotic cells were generated with fused ER and Golgi by brefeldin A treatment. Upon brefeldin A removal, Golgi vesicles did not emerge from the ER. In contrast, the Golgi readily reformed from similarly treated interphase cells. We conclude that Golgi-derived vesicles remain distinct from the ER in mitotic HeLa cells, and that mitotic cells lack the capacity of interphase cells for Golgi reemergence from the ER. These experiments suggest that mitotic Golgi breakdown proceeds by direct vesiculation independent of the ER.     

A protein kinase, PKN, accumulates in Alzheimer neurofibrillary tangles and associated endoplasmic reticulum-derived vesicles and phosphorylates tau protein

A possible role for a protein kinase, PKN, a fatty acid-activated serine/threonine kinase with a catalytic domain homologous to the protein kinase C family and a direct target for Rho, was investigated in the pathology of Alzheimer's disease (AD) using a sensitive immunocytochemistry on postmortem human brain tissues and a kinase assay for human tau protein. The present study provides evidences by light, electron, and confocal laser microscopy that in control human brains, PKN is enriched in neurons, where the kinase is concentrated in a subset of endoplasmic reticulum (ER) and ER-derived vesicles localized to the apical compartment of juxtanuclear cytoplasm, as well as late endosomes, multivesicular bodies, Golgi bodies, secretary vesicles, and nuclei. In AD-affected neurons, PKN was redistributed to the cortical cytoplasm and neurites and was closely associated with neurofibrillary tangles (NFTs) and their major constituent, abnormally modified tau. PKN was also found in degenerative neurites within senile plaques. In addition, we report that human tau protein is directly phosphorylated by PKN both in vitro and in vivo. Thus, our results suggest a specific role for PKN in NFT formation and neurodegeneration in AD damaged neurons.     

GPI anchor attachment is required for Gas1p transport from the endoplasmic reticulum in COP II vesicles

Inositol starvation of auxotrophic yeast interrupts glycolipid biosynthesis and prevents lipid modification of a normally glycosyl phosphatidylinositol (GPI)-linked protein, Gas1p. The unanchored Gas1p precursor undergoes progressive modification in the endoplasmic reticulum (ER), but is not modified by Golgi-specific glycosylation. Starvation-induced defects in anchor assembly and protein processing are rapid, and occur without altered maturation of other proteins. Cells remain competent to manufacture anchor components and to process Gas1p efficiently once inositol is restored. Newly synthesized Gas1p is packaged into vesicles formed in vitro from perforated yeast spheroplasts incubated with either yeast cytosol or the purified Sec proteins (COP II) required for vesicle budding from the ER. In vitro synthesized vesicles produced by inositol-starved membranes do not contain detectable Gas1p. These studies demonstrate that COP II components fulfill the soluble protein requirements for packaging a GPI-anchored protein into ER-derived transport vesicles. However, GPI anchor attachment is required for this packaging to occur.     

A novel muscle protein located inside the terminal cisternae of the sarcoplasmic reticulum

An immunofluorescence study of adult rat muscle tissues with a polyclonal antibody against the RGD-directed fibronectin receptor of Friend's erythroleukemia cells (alpha5beta1-integrin) unexpectedly revealed a pattern of intracellular antigen distribution. Western blotting analysis of rat and rabbit membrane fractions indicated that the antibody recognizes a 167-kDa protein expressed both in heart and in skeletal muscle (relative abundance: heart > slow muscle > fast muscle), but not in liver and kidney. The 167-kDa protein did not show altered electrophoretic mobility upon reduction and failed to bind several lectins, including wheat germ agglutinin. A study of its subcellular distribution in rabbit skeletal muscle revealed that the 167-kDa protein is mostly associated with the terminal cisternae of the sarcoplasmic reticulum (SR) and, to a smaller extent, with the sarcolemma, while it is absent in the longitudinal tubules of the SR. The 167-kDa protein is not an integral membrane protein since it can be extracted at pH >/=10. This protein can be proteolytically cleaved only in the presence of detergent, indicating that it resides on the luminal side of the SR. The 167-kDa protein could be resolved from the closely spaced sarcalumenin and histidine-rich protein by column chromatography followed by detergent dialysis and two-dimensional gel electrophoresis. The N terminus and the internal sequences did not match any known sequence in protein and DNA data bases, indicating that the 167-kDa protein is a novel muscle protein selectively localized to the SR. Integrins from rat kidney fibroblasts were not recognized by either (i) a polyclonal antiserum against the purified 167-kDa protein or (ii) the anti-alpha5beta1-integrin antiserum after affinity purification onto the 167-kDa protein. These data indicate that the 167-kDa protein is not immunologically cross-reactive with integrins, despite its reaction with a polyclonal anti-integrin antibody.     

Targeting to the endoplasmic reticulum in yeast cells by determinants present in transmembrane domains

The transmembrane domains (TMDs) of many type I integral membrane proteins contain determinants that cause localization in the endoplasmic reticulum (ER) in mammalian cells by an unknown mechanism. Here we show that the yeast ER localization machinery recognizes determinants in TMDs that are very similar to those identified previously in mammalian cells. These determinants are recognized in post-ER compartments and recycled back to the ER, thus acting as ER retrieval signals. Moreover determinants in TMDs are inefficiently sorted in several previously characterized yeast mutants with defects in the ER retrieval machinery. Similar ER retrieval signals are also recognized in the TMDs of polytopic integral membrane proteins, apparently by the same sorting machinery. The isolation of new mutants defective in sorting of membrane determinants might provide a better understanding of the molecular mechanisms involved in this process.     

Cloning and characterization of a novel zinc finger protein that associates with nuclear matrix

We have recently reported that the nuclei of B16 melanoma cells are intensely stained with anti-rat vitronectin (Vn) antibody, which reacts with both mouse and rat Vn. In the present study, we characterized the protein immunoreactive with the antibody using NIH3T3 cells, whose nuclei were also stained with the antibody. Western blot analysis showed that a protein with an approximate molecular weight of 75 kDa (p75), which was distinct from Vn, existed in the nuclear fraction, and, more specifically, in the nuclear matrix fraction, of NIH3T3 cells. Screening of an NIH3T3 cDNA library resulted in the isolation of a nearly full-length cDNA clone encoding p75. A database search revealed that the cDNA represents a novel gene. The deduced amino acid sequence showed that the protein is 580 amino acids long and contains two C2H2-type zinc finger motifs and glutamic acid-rich domains in the C-terminal region. When a fusion protein of green fluorescence protein and p75 was expressed in NIH3T3 cells, fluorescence was preferentially observed in the nuclei, demonstrating that the protein has a nuclear localization signal. The p75 protein, termed ZAN75, exhibited DNA-binding activity in a zinc-dependent manner. Southern blot analysis demonstrated that the ZAN75 gene exists in a single copy in the mouse genome and that a closely related gene is also present in chicken, rat, and human. Northern blot analysis showed that the ZAN75 gene is ubiquitously expressed in adult mouse tissues. In the cell cycle of NIH3T3 cells, expression was low in the G0/G1 phase, increased during the G1 phase, and persisted during the S and G2/M phases, suggesting that ZAN75 plays a role in regulating cell growth.