The 70 carboxyl-terminal amino acids of nascent secretory proteins are protected from proteolysis by the ribosome and the protein translocation apparatus of the endoplasmic reticulum membrane

We have used proteolysis to examine the environment through which nascent secretory proteins are translocated across the membrane of the endoplasmic reticulum. After solubilization of rough microsomes with detergent, fragments comprised of the approximately 70 carboxyl-terminal amino acids of translocating nascent chains initiated and targeted in vivo were protected from digestion by added proteases. About 40 amino acids of nascent chains were protected from proteolysis by the ribosome; thus, membrane-derived components protect an additional 30 amino acids. Under conditions in which those 30 additional amino acids are protected, only a small set of integral membrane proteins remained associated with the ribosome. These proteins include the Sec61 complex previously identified as the core component of the membrane-bound protein translocation apparatus. These results support the concept of a translocation pore that makes intimate contact with the ribosome and thereby protects nascent chains from proteolytic digestion for an additional, constant length.     

Sec61p serves multiple roles in secretory precursor binding and translocation into the endoplasmic reticulum membrane

The evolutionarily conserved Sec61 protein complex mediates the translocation of secretory proteins into the endoplasmic reticulum. To investigate the role of Sec61p, which is the main subunit of this complex, we generated recessive, cold-sensitive alleles of sec61 that encode stably expressed proteins with strong defects in translocation. The stage at which posttranslational translocation was blocked was probed by chemical crosslinking of radiolabeled secretory precursors added to membranes isolated from wild-type and mutant strains. Two classes of sec61 mutants were distinguished. The first class of mutants was defective in preprotein docking onto a receptor site of the translocon that included Sec61p itself. The second class of mutants allowed docking of precursors onto the translocon but was defective in the ATP-dependent release of precursors from this site that in wild-type membranes leads to pore insertion and full translocation. Only mutants of the second class were partially suppressed by overexpression of SEC63, which encodes a subunit of the Sec61 holoenzyme complex responsible for positioning Kar2p (yeast BiP) at the translocation channel. These mutants thus define two early stages of translocation that require SEC61 function before precursor protein transfer across the endoplasmic reticulum membrane.     

Nordihydroguaiaretic acid blocks protein transport in the secretory pathway causing redistribution of Golgi proteins into the endoplasmic reticulum

We have investigated the effect of nordihydroguaiaretic acid (NDGA), an inhibitor of lipoxygenase, on the intracellular protein transport and the structure of the Golgi complex. Pulse-chase experiments and immunoelectron microscopy showed that NDGA strongly inhibits the transport of newly synthesized secretory proteins to the Golgi complex resulting in their accumulation in the endoplasmic reticulum (ER). Despite their retention in the ER, oligosaccharides of secretory and ER-resident proteins were processed to endoglycosidase H-resistant forms, raising the possibility that oligosaccharide-processing enzymes are redistributed from the Golgi to the ER. Morphological observations further revealed that alpha-mannosidase II (a cis/medial-Golgi marker), but not TGN38 (a trans-Golgi network marker), rapidly redistributes to the ER in the presence of NDGA, resulting in the disappearance of the characteristic Golgi structure. Upon removal of the drug, the Golgi complex was reassembled into the normal structure as judged by perinuclear staining of alpha-mannosidase II and by restoration of the secretion. These effects of NDGA are quite similar to those of brefeldin A. However, unlike brefeldin A, NDGA did not cause a dissociation of beta-coatomer protein, a subunit of coatomer, from the Golgi membrane. On the contrary, NDGA exerted the stabilizing effect on beta-coatomer protein/membrane interaction against the dissociation caused by brefeldin A and ATP depletion. Taken together, these results indicate that NDGA is a potent agent disrupting the structure and function of the Golgi complex with a mechanism different from those known for other drugs reported so far.     

Detection of transient in vivo interactions between substrate and transporter during protein translocation into the endoplasmic reticulum

The split-ubiquitin technique was used to detect transient protein interactions in living cells. Nub, the N-terminal half of ubiquitin (Ub), was fused to Sec62p, a component of the protein translocation machinery in the endoplasmic reticulum of Saccharomyces cerevisiae. Cub, the C-terminal half of Ub, was fused to the C terminus of a signal sequence. The reconstitution of a quasi-native Ub structure from the two halves of Ub, and the resulting cleavage by Ub-specific proteases at the C terminus of Cub, serve as a gauge of proximity between the two test proteins linked to Nub and Cub. Using this assay, we show that Sec62p is spatially close to the signal sequence of the prepro-alpha-factor in vivo. This proximity is confined to the nascent polypeptide chain immediately following the signal sequence. In addition, the extent of proximity depends on the nature of the signal sequence. Cub fusions that bore the signal sequence of invertase resulted in a much lower Ub reconstitution with Nub-Sec62p than otherwise identical test proteins bearing the signal sequence of prepro-alpha-factor. An inactive derivative of Sec62p failed to interact with signal sequences in this assay. These in vivo findings are consistent with Sec62p being part of a signal sequence-binding complex.     

Folding of active beta-lactamase in the yeast cytoplasm before translocation into the endoplasmic reticulum

Polypeptides targeted to the yeast endoplasmic reticulum (ER) posttranslationally are thought to be kept in the cytoplasm in an unfolded state by Hsp70 chaperones before translocation. We show here that Escherichia coli beta-lactamase associated with Hsp70, but adopted a native-like conformation before translocation in living Saccharomyces cerevisiae cells. beta-Lactamase is a globular trypsin-resistant molecule in authentic form. For these studies, it was linked to the C terminus of a yeast polypeptide Hsp150delta, which conferred posttranslational translocation and provided sites for O-glycosylation. We devised conditions to retard translocation of Hsp150delta-beta-lactamase. This enabled us to show by protease protection assays that an unglycosylated precursor was associated with the cytoplasmic surface of isolated microsomes, whereas a glycosylated form resided inside the vesicles. Both proteins were trypsin resistant and had similar beta-lactamase activity and Km values for nitrocefin. The enzymatically active cytoplasmic intermediate could be chased into the ER, followed by secretion of the activity to the medium. Productive folding in the cytoplasm occurred in the absence of disulfide formation, whereas in the ER lumen, proper folding required oxidation of the sulfhydryls. This suggests that the polypeptide was refolded in the ER and consequently, at least partially unfolded for translocation.     

Redox state of single chain Fv fragments targeted to the endoplasmic reticulum, cytosol and mitochondria

In this paper we have engineered the targeting of ScFv fragments to mitochondria and demonstrated that this can occur efficiently. This extends the range of subcellular compartments where antibody domains can be targeted in order to interfere with the action of the corresponding antigen. Moreover, we have compared the redox state of ScFv fragments targeted to the secretory compartment, the cytosol and the mitochondria, and demonstrated that cysteine residues in ScFv targeted to the secretory compartments and to the mitochondria are oxidized. On the contrary, cytosolic antibody domains are expressed in a reduced state, which is probably the reason for their lower expression levels. These pitfalls, however, do not prevent their successful utilization for intracellular immunization.     

Ubiquitination is required for the retro-translocation of a short-lived luminal endoplasmic reticulum glycoprotein to the cytosol for degradation by the proteasome

In the endoplasmic reticulum (ER), an efficient "quality control system" operates to ensure that mutated and incorrectly folded proteins are selectively degraded. We are studying ER-associated degradation using a truncated variant of the rough ER-specific type I transmembrane glycoprotein, ribophorin I. The truncated polypeptide (RI332) consists of only the 332 amino-terminal amino acids of the protein corresponding to most of its luminal domain and, in contrast to the long-lived endogenous ribophorin I, is rapidly degraded. Here we show that the ubiquitin-proteasome pathway is involved in the destruction of the truncated ribophorin I. Thus, when RI332 that itself appears to be a substrate for ubiquitination was expressed in a mutant hamster cell line harboring a temperature-sensitive mutation in the ubiquitin-activating enzyme E1 affecting ubiquitin-dependent proteolysis, the protein is dramatically stabilized at the restrictive temperature. Moreover, inhibitors of proteasome function effectively block the degradation of RI332. Cell fractionation experiments indicate that RI332 accumulates in the cytosol when degradation is prevented by proteasome inhibitors but remains associated with the lumen of the ER under ubiquitination-deficient conditions, suggesting that the release of the protein into the cytosol is ubiquitination-dependent. Accordingly, when ubiquitination is impaired, a considerable amount of RI332 binds to the ER chaperone calnexin and to the Sec61 complex that could effect retro-translocation of the polypeptide to the cytosol. Before proteolysis of RI332, its N-linked oligosaccharide is cleaved in two distinct steps, the first of which might occur when the protein is still associated with the ER, as the trimmed glycoprotein intermediate efficiently interacts with calnexin and Sec61. From our data we conclude that the steps that lead a newly synthesized luminal ER glycoprotein to degradation by the proteasome are tightly coupled and that especially ubiquitination plays a crucial role in the retro-translocation of the substrate protein for proteolysis to the cytosol.     

Peroxisome biogenesis: back to the endoplasmic reticulum?

Proteins are targeted to the membrane and matrix of peroxisomes by distinct pathways. Recent observations suggest a further route: a subset of peroxisomal membrane proteins might be targeted first to the endoplasmic reticulum, and from there to peroxisomes by vesicle-mediated transport.     

The Alzheimer's disease-associated presenilins are differentially phosphorylated proteins located predominantly within the endoplasmic reticulum

BACKGROUND: Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by the deposition of extracellular senile plaques composed of amyloid beta-peptide (A beta). Whereas most cases of AD occur sporadically, about 10% of AD cases are inherited as a fully penetrant autosomal dominant trait. Mutations in the recently cloned Presenilin genes (PS-1 and PS-2) are by far the most common cause of early onset familial AD. MATERIALS AND METHODS: Cellular expression of endogenous and overexpressed PS proteins was analyzed by immunocytochemistry and metabolic labeling followed by immunoprecipitation. In vivo phosphorylation sites of PS proteins were analyzed by extensive mutagenesis. RESULTS: PS-1 as well as PS-2 proteins were localized predominantly within the endoplasmic reticulum (ER). However, small amounts of the PS proteins were detected within the Golgi compartment, where they colocalize with the beta-amyloid precursor protein (beta APP). The PS-2 protein was found to be highly phosphorylated, whereas very little phosphorylation was observed for PS-1. The selective phosphorylation of PS-2 occurs exclusively on serine residues. In vivo phosphorylation of PS-2 was mapped to serine residues 7, 9, and 19 within an acidic stretch at the N terminus, which is absent in PS-1. casein kinase (CK)-1 and CK-2 were shown to phosphorylate the N terminus of PS-2 in vitro. CONCLUSIONS: The majority of PS proteins were detected in the ER where little if any proteolytic processing of beta APP was reported. ER retention of PS proteins might occur by intramolecular aggregation. Small amounts of PS proteins were also detected in the Golgi where they colocalized with beta APP. This might suggest that potential interactions between PS proteins and beta APP could occur within the Golgi. Selective phosphorylation of PS-2 proteins within the acidic domain missing in PS-1 indicates differences in the biological functions and regulation of the two highly homologous proteins.     

Protein disulfide isomerase is the dominant acceptor for peptides translocated into the endoplasmic reticulum

Peptides derived from cytosolic protein degradation are translocated into the lumen of the endoplasmic reticulum (ER) by the transporter associated with antigen processing (TAP). In the ER, class I molecules bind the peptides fitting to their respective motifs and present them on the cell surface to CD8+ T lymphocytes. However, most TAP-translocated peptides are not expected to bind to the class I molecules present in a particular cell. Recently, we have demonstrated that TAP-translocated peptides containing a photoreactive phenylalanine analogue can be cross-linked to two luminal ER-resident proteins: with low efficiency to the stress protein gp96 and with high efficiency to a 60-kDa protein (Lammert, E. et al., Eur. J. Immunol. 1997. 27: 923). Both proteins have also been labeled specifically by TAP-translocated peptides conjugated to a different photoreactive group (Marusina, K. et al., Biochemistry 1997. 36: 856). Here, we show that the 60-kDa peptide-binding protein is identical to the multifunctional protein disulfide isomerase (PDI). Since PDI is the only luminal ER-resident protein that is labeled by the photoreactive peptides with high efficiency, it might represent the dominant acceptor for TAP-translocated peptides.     

Determination of the transmembrane topology of yeast Sec61p, an essential component of the endoplasmic reticulum translocation complex

Sec61p is a highly conserved integral membrane protein that plays a role in the formation of a protein-conducting channel required for the translocation of polypeptides into, and across, the membrane of the endoplasmic reticulum. As a major step toward elucidating the structure of the endoplasmic reticulum translocation apparatus, we have determined the transmembrane topology of Sec61p using a combination of C-terminal reporter-domain fusions and the in situ digestion of specifically inserted factor Xa protease cleavage sites. Our data indicate the presence of 10 transmembrane domains, including several with surprisingly limited hydrophobicity. Furthermore, we provide evidence for complex intramolecular interactions in which these weakly hydrophobic domains require C-terminal sequences for their correct topogenesis. The incorporation of sequences with limited hydrophobicity into the bilayer may play a vital role in the formation of an aqueous membrane channel required for the translocation of hydrophilic polypeptide chains.     

Noninvasive measurement of the pH of the endoplasmic reticulum at rest and during calcium release

The pH within individual organelles of the secretory pathway is believed to be an important determinant of their biosynthetic activity. However, little is known about the determinants and regulation of the pH in the secretory organelles, which cannot be readily accessed by [H+]-sensitive probes. We devised a procedure for the dynamic, noninvasive measurement of pH in the lumen of the endoplasmic reticulum in intact mammalian cells. A recombinant form of the B subunit of Shiga toxin, previously modified to include a carboxyl-terminal KDEL sequence and a pH-sensitive fluorophore, was used for a two-stage delivery strategy. Retrograde traffic of endogenous lipids was harnessed to target this protein to the Golgi complex, followed by retrieval to the endoplasmic reticulum (ER) by KDEL receptors. Immunofluorescence and immunoelectron microscopy were used to verify the subcellular localization of the modified B fragment. Fluorescence ratio imaging and two independent calibration procedures were applied to determine the pH of the ER in situ. We found that the pH of the endoplasmic reticulum is near neutral and is unaffected during agonist-induced release of calcium. The ER was found to be highly permeable to H+ (equivalents), so that the prevailing [H+] is susceptible to alterations in the cytosolic pH. Plasmalemmal acid-base transporters were shown to indirectly regulate the endoplasmic reticulum pH.     

Structural changes in the endoplasmic reticulum of starfish oocytes during meiotic maturation and fertilization

The endoplasmic reticulum (ER) of live starfish oocytes was observed during meiotic maturation and fertilization. The ER was visualized by injection into the cytoplasm of an oil drop saturated with the fluorescent lipophilic dye DiI; DiI spread throughout the oocyte endoplasmic reticulum and the pattern was imaged by confocal microscopy. The ER in the immature (germinal vesicle stage) oocyte was composed of interconnected membrane sheets. In response to 1-methyladenine, the sheets of ER appeared to become associated with the yolk platelets, forming spherical shells. A few of these spherical shells could sometimes be seen in immature oocytes, but their number was much greater in the egg at the first meiotic spindle stage. At about the time that the first polar body formed, the spherical shells disappeared, and the ER returned to a form like that of the immature oocyte. The spherical shells did not reappear during the second meiotic cycle. During maturation, the ER also began to move; the movement was apparent by the time of germinal vesicle breakdown and continued throughout both meiotic cycles and in eggs with second polar bodies. When eggs at the first meiotic spindle stage were fertilized, the form of the ER changed. Within 1 min after sperm addition to the observation chamber, the circular cross sections of the spherical shells of the unfertilized egg ER were no longer distinct. At this point, the form of the ER could not be discerned with the resolution of the light microscope; however, the rate of spreading of DiI from an injected oil drop decreased, providing strong evidence that the ER had become fragmented. The ER remained in this form for several minutes and then gradually, the appearance of the ER and the rate of DiI spreading returned to be like those of the unfertilized egg. Injection of inositol trisphosphate caused a similar change in the ER structure. These results indicate that the ER is a dynamic structure, the form of which changes during oocyte maturation and fertilization.     

Targeting of Yersinia Yop proteins into the cytosol of HeLa cells: one-step translocation of YopE across bacterial and eukaryotic membranes is dependent on SycE chaperone

Pathogenic Yersiniae adhere to and kill macrophages by targeting some of their Yop proteins into the eukaryotic cytosol. There is debate about whether YopE targeting proceeds as a direct translocation of polypeptide between cells or in two distinct steps, each requiring specific signals for YopE secretion across the bacterial envelope and for translocation into the eukaryotic cytosol. Here, we used the selective solubilization of the eukaryotic plasma membrane with digitonin to measure Yop targeting during Yersinia infections of HeLa cells. YopE, YopH, YopM and YopN were found in the eukaryotic cytosol but not in the extracellular medium. When bound to SycE chaperone in the Yersinia cytoplasm, YopE residues 1-100 are necessary and sufficient for the targeting of hybrid neomycin phosphotransferase. Electron microscopic analysis failed to detect an extracellular intermediate of YopE targeting, suggesting a one-step translocation mechanism.     

The ontogeny of key endoplasmic reticulum proteins in human embryonic and fetal red blood cells

Recently, using immunohistochemical methods, we surprisingly found that endoplasmic reticulum glucose-6-phosphatase is present in human embryonic and fetal red blood cells (RBCs) but not in adult RBCs. The fact that an endoplasmic reticulum enzyme, whose major site of expression in adults is the liver, is present in human embryonic and fetal RBCs, particularly nucleated cells, indicated that it would be sensible to determine whether these cells also contain other endoplasmic reticulum enzyme systems normally found in adult liver. Therefore, we have studied the expression of other endoplasmic reticulum proteins and found that human embryonic and fetal RBC precursors contain other protein components of the glucose-6-phosphatase system, ie, the phosphate and glucose transport proteins as well as other enzymes (eg, uridine diphosphate-glucuronosyltransferases, cytochrome P450 isozymes, nicotinamide adenine dinucleotide phosphate cytochrome P450 oxidoreductase, and prostaglandin H synthase). In addition, we also found the predominantly cytosolic markers 15-hydroxyprostaglandin dehydrogenase, prostaglandins PGE2 and 13,14-dihydro-15-keto-PGE2. The expression of key enzymes that control glucose production, detoxification of endobiotics and xenobiotics, and the regulation of prostaglandin levels in embryonic and early fetal RBCs means that these cells may have an important role in protecting the developing conceptus before it establishes an efficient circulation and before all tissues fully express their normal complement of these enzymes.     

The relationship of the endoplasmic reticulum to the Golgi complex in Tetrahymena pyriformis

The endoplasmic reticulum (ER) and the closely connected, single dictyosomal Golgi apparatus of Tetrahymena pyriformis cells showed random distribution in the cytoplasm. Ribosomes were evident, and coated vesicles pinched off from the ER were seen. The membranes of the endoplasmic reticulum generally formed a tube-like structure, although after histamine treatment multiple, folded and circular structures were observed. The number of coated vesicles detaching from the endoplasmic reticulum increased as a result of histamine treatment.     

Evidence that endoplasmic reticulum (ER)-associated degradation of cystic fibrosis transmembrane conductance regulator is linked to retrograde translocation from the ER membrane

The ubiquitin-proteasome pathway has been implicated in the degradation of newly synthesized, misfolded and unassembled proteins in the endoplasmic reticulum (ER). Using a cell-free reticulocyte lysate system we have examined the relationship between biosynthesis and ER-associated degradation of the cystic fibrosis transmembrane conductance regulator (CFTR), a polytopic protein with 12 predicted transmembrane segments. Our results provide direct evidence that full-length, glycosylated and membrane-integrated CFTR is a substrate for degradation and that degradation involves polyubiquitination and cytosolic proteolytic activity. CFTR ubiquitination was both temperature- and ATP-dependent. Degradation was significantly inhibited by EDTA, apyrase, and the proteasome inhibitors hemin and MG132. Degradation was inhibited to a lesser extent by clasto-lactacystin beta-lactone, ALLN, and Nalpha-tosyl-L-phenylalanine chloromethyl ketone and was relatively unaffected by lactacystin and N-tosyl lysyl chloromethyl ketone. In the presence of hemin, polyubiquitinated CFTR remained tightly associated with ER microsomes. However, membrane-bound ubiquitinated CFTR could be subsequently degraded into trichloroacetic acid-soluble fragments upon incubation in hemin-free, ATP-containing lysate. Thus ER-associated degradation of CFTR occurs via a membrane-bound, rather than cytosolic, intermediate and likely involves recruitment of degradation machinery to the ER membrane. Our data suggest a model in which the degradation of polytopic proteins such as CFTR is coupled to retrograde translocation and removal of the polypeptide from the lipid bilayer.     

Changing patterns of localization of the tobacco mosaic virus movement protein and replicase to the endoplasmic reticulum and microtubules during infection

Tobacco mosaic virus (TMV) derivatives that encode movement protein (MP) as a fusion to the green fluorescent protein (MP:GFP) were used in combination with antibody staining to identify host cell components to which MP and replicase accumulate in cells of infected Nicotiana benthamiana leaves and in infected BY-2 protoplasts. MP:GFP and replicase colocalized to the endoplasmic reticulum (ER; especially the cortical ER) and were present in large, irregularly shaped, ER-derived structures that may represent "viral factories." The ER-derived structures required an intact cytoskeleton, and microtubules appeared to redistribute MP:GFP from these sites during late stages of infection. In leaves, MP:GFP accumulated in plasmodesmata, whereas in protoplasts, the MP:GFP was targeted to distinct, punctate sites near the plasma membrane. Treating protoplasts with cytochalasin D and brefeldin A at the time of inoculation prevented the accumulation of MP:GFP at these sites. It is proposed that the punctate sites anchor the cortical ER to plasma membrane and are related to sites at which plasmodesmata form in walled cells. Hairlike structures containing MP:GFP appeared on the surface of some of the infected protoplasts and are reminiscent of similar structures induced by other plant viruses. We present a model that postulates the role of the ER and cytoskeleton in targeting the MP and viral ribonucleoprotein from sites of virus synthesis to the plasmodesmata through which infection is spread.     

Intracellular translocation and stability of apolipoprotein B are inversely proportional to the length of the nascent polypeptide

We have studied the relationship between the length of apolipoprotein B (apoB) and its intracellular translocation and stability using McArdle RH7777 (McA-RH7777) cells expressing recombinant human apoB variants, ranging in size from B15 to B100. The translocational status of apoB was assessed based on trypsin sensitivity of apoB using isolated microsomes as well as permeabilized cells. In isolated microsomes, shorter apoB variants (相似文献