Segregation of the signal sequence receptor protein in the rough endoplasmic reticulum membrane

The signal sequence receptor (SSR), an integral membrane glycoprotein of 34 kDa, has previously been shown to be a component of the molecular environment which nascent polypeptide chains meet in passage through the endoplasmic reticulum (ER) membrane. We have used antibodies directed against the SSR and both immunocytochemistry and cell fractionation to determine its distribution in rat liver cells. SSR was found largely restricted to the rough ER. Only small amounts of the protein were detected in smooth ER. These results provide further evidence for a functional differentiation of rough and smooth ER and for a role of SSR in protein translocation across the ER membrane.     

The unfolded protein response coordinates the production of endoplasmic reticulum protein and endoplasmic reticulum membrane.

The endoplasmic reticulum (ER) is a multifunctional organelle responsible for production of both lumenal and membrane components of secretory pathway compartments. Secretory proteins are folded, processed, and sorted in the ER lumen and lipid synthesis occurs on the ER membrane itself. In the yeast Saccharomyces cerevisiae, synthesis of ER components is highly regulated: the ER-resident proteins by the unfolded protein response and membrane lipid synthesis by the inositol response. We demonstrate that these two responses are intimately linked, forming different branches of the same pathway. Furthermore, we present evidence indicating that this coordinate regulation plays a role in ER biogenesis.     

A defective signal peptide tethers the floury-2 zein to the endoplasmic reticulum membrane.

The maize (Zea mays L.) floury-2 (fl2) mutation is associated with a general decrease in storage protein synthesis, altered protein body morphology, and the synthesis of a novel 24-kD alpha-zein storage protein. Unlike storage proteins in normal kernels and the majority of storage proteins in fl2 kernels, the 24-kD alpha-zein contains a signal peptide that would normally be removed during protein synthesis and processing. The expected processing site of this alpha-zein reveals a putative mutation alanine-->valine (Ala-->Val) that is not found at other junctions between signal sequences and mature proteins. To investigate the impact of such a mutation on signal peptide cleavage, we have assayed the 24-kD fl2 alpha-zein in a co-translational processing system in vitro. Translation of RNA from fl2 kernels or synthetic RNA encoding the fl2 alpha-zein in the presence of microsomes yielded a 24-kD polypeptide. A normal signal peptide sequence, generated by site-directed mutagenesis, restored the capacity of the RNA to direct synthesis of a properly processed protein in a cell-free system. Both the fl2 alpha-zein and the fl2 alpha-zein (Val-->Ala) were translocated into the lumen of the endoplasmic reticulum. The processed fl2 alpha-zein (Val-->Ala) was localized in the soluble portion of the microsomes, whereas the fl2 alpha-zein co-fractionated with the microsomal membranes. By remaining anchored to protein body membranes during endosperm maturation, the fl2 zein may thus constrain storage protein packing and perturb protein body morphology.     

Interactions between the endoplasmic reticulum, mitochondria, plasma membrane and other subcellular organelles

Several recent works show structurally and functionally dynamic contacts between mitochondria, the plasma membrane, the endoplasmic reticulum, and other subcellular organelles. Many cellular processes require proper cooperation between the plasma membrane, the nucleus and subcellular vesicular/tubular networks such as mitochondria and the endoplasmic reticulum. It has been suggested that such contacts are crucial for the synthesis and intracellular transport of phospholipids as well as for intracellular Ca²⁺ homeostasis, controlling fundamental processes like motility and contraction, secretion, cell growth, proliferation and apoptosis. Close contacts between smooth sub-domains of the endoplasmic reticulum and mitochondria have been shown to be required also for maintaining mitochondrial structure. The overall distance between the associating organelle membranes as quantified by electron microscopy is small enough to allow contact formation by proteins present on their surfaces, allowing and regulating their interactions. In this review we give a historical overview of studies on organelle interactions, and summarize the present knowledge and hypotheses concerning their regulation and (patho)physiological consequences.     

Infectious bronchitis virus 3a protein localizes to a novel domain of the smooth endoplasmic reticulum

All coronaviruses possess small open reading frames (ORFs) between structural genes that have been hypothesized to play important roles in pathogenesis. Infectious bronchitis virus (IBV) ORF 3a is one such gene. It is highly conserved among group 3 coronaviruses, suggesting that it has an important function in infection. IBV 3a protein is expressed in infected cells but is not detected in virions. Sequence analysis predicted that IBV 3a was a membrane protein; however, only a fraction behaved like an integral membrane protein. Microscopy and immunoprecipitation studies demonstrated that IBV 3a localized to the cytoplasm in a diffuse pattern as well as in sharp puncta in both infected and transfected cells. These puncta did not overlap cellular organelles or other punctate structures. Confocal microscopy demonstrated that IBV 3a puncta lined up along smooth endoplasmic reticulum (ER) tubules and, in a significant number of instances, were partially surrounded by these tubules. Our results suggest that IBV 3a is partially targeted to a novel domain of the smooth ER.     

Topology and functional domains of Sec63p, an endoplasmic reticulum membrane protein required for secretory protein translocation.

SEC63 encodes a protein required for secretory protein translocation into the endoplasmic reticulum (ER) of Saccharomyces cerevisiae (J. A. Rothblatt, R. J. Deshaies, S. L. Sanders, G. Daum, and R. Schekman, J. Cell Biol. 109:2641-2652, 1989). Antibody directed against a recombinant form of the protein detects a 73-kDa polypeptide which, by immunofluorescence microscopy, is localized to the nuclear envelope-ER network. Cell fractionation and protease protection experiments confirm the prediction that Sec63p is an integral membrane protein. A series of SEC63-SUC2 fusion genes was created to assess the topology of Sec63p within the ER membrane. The largest hybrid proteins are unglycosylated, suggesting that the carboxyl terminus of Sec63p faces the cytosol. Invertase fusion to a loop in Sec63p that is flanked by two putative transmembrane domains produces an extensively glycosylated hybrid protein. This loop, which is homologous to the amino terminus of the Escherichia coli heat shock protein, DnaJ, is likely to face the ER lumen. By analogy to the interaction of the DnaJ and Hsp70-like DnaK proteins in E. coli, the DnaJ loop of Sec63p may recruit luminal Hsp70 (BiP/GRP78/Kar2p) to the translocation apparatus. Mutations in two highly conserved positions of the DnaJ loop and short deletions of the carboxyl terminus inactivate Sec63p activity. Sec63p associates with several other proteins, including Sec61p, a 31.5-kDa glycoprotein, and a 23-kDa protein, and together with these proteins may constitute part of the polypeptide translocation apparatus. A nonfunctional DnaJ domain mutant allele does not interfere with the formation of the Sec63p/Sec61p/gp31.5/p23 complex.     

Characterization of secretory protein translocation: ribosome-membrane interaction in endoplasmic reticulum

Secretory proteins are synthesized on ribosomes bound to the membrane of the endoplasmic reticulum (ER). After the selection of polysomes synthesizing secretory proteins and their direction to the membrane of the ER via signal recognition particle (SRP) and docking protein respectively, the polysomes become bound to the ER membrane via an unknown, protein-mediated mechanism. To identify proteins involved in protein translocation, beyond the (SRP-docking protein-mediated) recognition step, controlled proteolysis was used to functionally inactivate rough microsomes that had previously been depleted of docking protein. As the membranes were treated with increasing levels of protease, they lost their ability to be functionally reconstituted with the active cytoplasmic fragment of docking protein (DPf). This functional inactivation did not correlate with a loss of either signal peptidase activity, nor with the ability of the DPf to reassociate with the membrane. It did correlate, however, with a loss of the ability of the microsomes to bind ribosomes. Ribophorins are putative ribosome-binding proteins. Immunoblots developed with monoclonal antibodies against canine ribophorins I and II demonstrated that no correlation exists between the protease-induced inability to bind ribosomes and the integrity of the ribophorins. Ribophorin I was 85% resistant and ribophorin II 100% resistant to the levels of protease needed to totally eliminate ribosome binding. Moreover, no direct association was found between ribophorins and ribosomes; upon detergent solubilization at low salt concentrations, ribophorins could be sedimented in the presence or absence of ribosomes. Finally, the alkylating agent N-ethylmaleimide was shown to be capable of inhibiting translocation (beyond the SRP-docking protein-mediated recognition step), but had no affect on the ability of ribosomes to bind to ER membranes. We conclude that potentially two additional proteinaceous components, as yet unidentified, are involved in protein translocation. One is protease sensitive and possibly involved in ribosome binding, the other is N-ethylmaleimide sensitive and of unknown function.     

Topology and endoplasmic reticulum retention signals of the lysosomal storage disease-related membrane protein CLN6

CLN6 is a polytopic membrane protein of unknown function resident in the endoplasmic reticulum (ER). Mutant CLN6 causes the lysosomal storage disorder neuronal ceroid lipofuscinosis. Defining the topology of CLN6, and the structural domains and motifs required for interaction with cytosolic and luminal proteins may allow insights into its function. In this study we analysed the topology, ER retention and oligomerization of CLN6. We demonstrated, by differential membrane permeabilization of transfected BHK cells using specific detergents and two distinct antibodies, that CLN6 contains an N-terminal cytoplasmic domain, seven transmembrane domains, and a luminal C terminus. Mutational analyses and confocal immunofluorescence microscopy showed that changes of potential ER localization signals in the N- or C-terminal domain (a triple arginine cluster, and a dileucine motif) did not alter the subcellular localization of CLN6. The deletion of a dilysine motif impaired partially the ER localization of CLN6. Furthermore, expression analyses of fusion and deletion constructs in non-neuronal and neuronal cells suggested that two portions of CLN6 contributed to its retention within the ER. We showed that the N-terminal domain was necessary but not sufficient for ER retention of CLN6 and that deletion of transmembrane domains 6 and 7 was accompanied with the loss of ER localization and, in some instances, trafficking to the cisGolgi. From these data we concluded that CLN6 maintains its ER localization by expressing retention signals present in both the N-terminal cytosolic domain and in the carboxy-proximal transmembrane domains 6 and 7. Additionally, the ability of CLN6 to homodimerize may also prevent exit from the ER via an interaction with membrane-associated factors.     

An ATP-binding membrane protein is required for protein translocation across the endoplasmic reticulum membrane.

The role of nucleotides in providing energy for polypeptide transfer across the endoplasmic reticulum (ER) membrane is still unknown. To address this question, we treated ER-derived mammalian microsomal vesicles with a photoactivatable analogue of ATP, 8-N3ATP. This treatment resulted in a progressive inhibition of translocation activity. Approximately 20 microsomal membrane proteins were labeled by [alpha 32P]8-N3ATP. Two of these were identified as proteins with putative roles in translocation, alpha signal sequence receptor (SSR), the 35-kDa subunit of the signal sequence receptor complex, and ER-p180, a putative ribosome receptor. We found that there was a positive correlation between inactivation of translocation activity and photolabeling of alpha SSR. In contrast, our data demonstrate that the ATP-binding domain of ER-p180 is dispensable for translocation activity and does not contribute to the observed 8-N3ATP sensitivity of the microsomal vesicles.     

Posttranslational protein translocation across the membrane of the endoplasmic reticulum

Posttranslational protein translocation across the membrane of the endoplasmic reticulum is mediated by the Sec complex. This complex includes a transmembrane channel formed by multiple copies of the Sec61 protein. Translocation of a polypeptide begins when the signal sequence binds at a specific site within the channel. Binding results in the insertion of the substrate into the channel, possibly as a loop with a small segment exposed to the lumen. While bound, the signal sequence is in contact with both protein components of the channel and the lipid of the membrane. Subsequent movement of the polypeptide through the channel occurs when BiP molecules interact transiently with a luminal domain of the Sec complex, hydrolyze ATP, and bind to the substrate. Bound BiP promotes translocation by preventing the substrate from diffusing backwards through the channel, and thus acts as a molecular ratchet.     

Insertion and topology of a plant viral movement protein in the endoplasmic reticulum membrane

Virus-encoded movement proteins (MPs) mediate cell-to-cell spread of viral RNA through plant membranous intercellular connections, the plasmodesmata. The molecular pathway by which MPs interact with viral genomes and target plasmodesmata channels is largely unknown. The 9-kDa MP from carnation mottle carmovirus (CarMV) contains two potential transmembrane domains. To explore the possibility that this protein is in fact an intrinsic membrane protein, we have investigated its insertion into the endoplasmic reticulum membrane. By using in vitro translation in the presence of dog pancreas microsomes, we demonstrate that CarMV p9 inserts into the endoplasmic reticulum without the aid of any additional viral or plant host components. We further show that the membrane topology of CarMV p9 is N(cyt)-C(cyt) (N and C termini of the protein facing the cytoplasm) by in vitro translation of a series of truncated and full-length constructs with engineered glycosylation sites. Based on these results, we propose a topological model in which CarMV p9 is anchored in the membrane with its N- and C-terminal tail segments interacting with its soluble, RNA-bound partner CarMV p7, to accomplish the viral cell-to-cell movement function.     

Acyl-CoA-binding protein (ACBP) localizes to the endoplasmic reticulum and Golgi in a ligand-dependent manner in mammalian cells

In the present study, we microinjected fluorescently labelled liver bovine ACBP (acyl-CoA-binding protein) [FACI-50 (fluorescent acyl-CoA indicator-50)] into HeLa and BMGE (bovine mammary gland epithelial) cell lines to characterize the localization and dynamics of ACBP in living cells. Results showed that ACBP targeted to the ER (endoplasmic reticulum) and Golgi in a ligand-binding-dependent manner. A variant Y28F/K32A-FACI-50, which is unable to bind acyl-CoA, did no longer show association with the ER and became segregated from the Golgi, as analysed by intensity correlation calculations. Depletion of fatty acids from cells by addition of FAFBSA (fatty-acid-free BSA) significantly decreased FACI-50 association with the Golgi, whereas fatty acid overloading increased Golgi association, strongly supporting that ACBP associates with the Golgi in a ligand-dependent manner. FRAP (fluorescence recovery after photobleaching) showed that the fatty-acid-induced targeting of FACI-50 to the Golgi resulted in a 5-fold reduction in FACI-50 mobility. We suggest that ACBP is targeted to the ER and Golgi in a ligand-binding-dependent manner in living cells and propose that ACBP may be involved in vesicular trafficking.