Reversible interruption of Giardia lamblia cyst wall protein transport in a novel regulated secretory pathway

To survive in the environment and infect a new host, Giardia lamblia secretes an extracellular cyst wall using a poorly understood pathway. The two cyst wall proteins (CWPs) form disulphide-bonded heterodimers and are exported via novel encystation-specific secretory vesicles (ESVs). Exposure of eukaryotic cells to dithiothreitol (DTT) blocks the formation of disulphide bonds in nascent proteins that accumulate in the endoplasmic reticulum (ER) and induces an unfolded protein response (UPR). Proteins that have exited the ER are not susceptible. Exposure to DTT inhibits ESV formation by > 85%. Addition of DTT to encysting cells causes rapid ( t _1/2 < 10 min), reversible disappearance of ESVs, correlated with reduction of CWPs to monomers and reformation of CWP oligomers upon removal of DTT. Neither CWPs nor ESVs are affected by mercaptoethanesulphonic acid, a strong reducing agent that does not penetrate cells. DTT does not inhibit the overall protein secretory pathway, and recovery does not require new protein synthesis. We found evidence of protein disulphide isomerases in the ESV and the surface of encysting cells, in which they may catalyse initial CWP folding and recovery from DTT. This is the first suggestion of non-CWP proteins in ESVs and of enzymes on the giardial surface. DTT treatment did not stimulate a UPR, suggesting that Giardia may have diverged before the advent of this conserved form of ER quality control.     

Genomic analysis of the unfolded protein response in Arabidopsis shows its connection to important cellular processes

We analyzed the breadth of the unfolded protein response (UPR) in Arabidopsis using gene expression analysis with Affymetrix GeneChips. With tunicamycin and DTT as endoplasmic reticulum (ER) stress-inducing agents, we identified sets of UPR genes that were induced or repressed by both stresses. The proteins encoded by most of the upregulated genes function as part of the secretory system and comprise chaperones, vesicle transport proteins, and ER-associated degradation proteins. Most of the downregulated genes encode extracellular proteins. Therefore, the UPR may constitute a triple effort by the cell: to improve protein folding and transport, to degrade unwanted proteins, and to allow fewer secretory proteins to enter the ER. No single consensus response element was found in the promoters of the 53 UPR upregulated genes, but half of the genes contained response elements also found in mammalian UPR regulated genes. These elements are enriched from 4.5- to 15-fold in this upregulated gene set.     

Expression of OsBiP4 and OsBiP5 is highly correlated with the endoplasmic reticulum stress response in rice

Binding protein (BiP) is a chaperone protein involved in the folding of secretory proteins in the ER lumen. OsBiP1 is constitutively expressed in various tissues, whereas the expression of OsBiP4 and OsBiP5 (OsBiP4&5) is not detected in any tissue under normal conditions. However, expression of OsBiP4&5 was highly and specifically activated under ER stress conditions induced by DTT treatment, OsBiP1 knockdown, OsBiP1 overexpression, OsIRE1 overexpression, or various exogenous recombinant proteins in transgenic rice. In contrast, OsBiP4&5 did not accumulate in OsIRE1 knockdown transgenic rice even after DTT treatment. When the subcellular localization of OsBiP4&5 was investigated in seed endosperm cells under the ER stress condition, OsBiP4&5 were localized to the ER, but did not participate in ER-derived protein body (PB-I) formation in a different manner to OsBiP1. These results indicate that OsBiP4&5 levels were positively correlated with stress levels in the ER. Taken together, these results suggest that OsBiP4&5 are ER stress-related BiP proteins that are regulated by OsIRE1/OsbZIP50 pathway and that they may have a distinct function from that of OsBiP1 in rice.     

植物表达分泌蛋白的运输及定位

分泌途径主要由内膜系统构成,内质网和高尔基体对于分泌蛋白的运输及定位具有重要作用。分泌蛋白的运输包括顺行途径和逆行途径。蛋白质通过质流和受体介导的途径运输到小泡中。在植物中,分泌蛋白的运输主要通过小泡和相连的小管来完成。分子伴侣和质量控制不仅能优化新合成蛋白的折叠和组装,而且去除了有折叠缺陷的蛋白。分泌蛋白的定位需要特定的信号肽,而高尔基体固有蛋白以依赖跨膜长度的方式,沿着分泌途径的细胞器分布。本文对植物表达分泌蛋白的分泌途径及定位、相关的分子伴侣和质量控制进行了综述。     

ER/Golgi intermediates acquire Golgi enzymes by brefeldin A-sensitive retrograde transport in vitro

Secretory proteins exit the ER in transport vesicles that fuse to form vesicular tubular clusters (VTCs) which move along microtubule tracks to the Golgi apparatus. Using the well-characterized in vitro approach to study the properties of Golgi membranes, we determined whether the Golgi enzyme NAGT I is transported to ER/Golgi intermediates. Secretory cargo was arrested at distinct steps of the secretory pathway of a glycosylation mutant cell line, and in vitro complementation of the glycosylation defect was determined. Complementation yield increased after ER exit of secretory cargo and was optimal when transport was blocked at an ER/Golgi intermediate step. The rapid drop of the complementation yield as secretory cargo progresses into the stack suggests that Golgi enzymes are preferentially targeted to ER/Golgi intermediates and not to membranes of the Golgi stack. Two mechanisms for in vitro complementation could be distinguished due to their different sensitivities to brefeldin A (BFA). Transport occurred either by direct fusion of preexisting transport intermediates with ER/Golgi intermediates, or it occurred as a BFA-sensitive and most likely COP I-mediated step. Direct fusion of ER/Golgi intermediates with cisternal membranes of the Golgi stack was not observed under these conditions.     

Disulfide bonds in folding and transport of mouse hepatitis coronavirus glycoproteins.

We have analyzed the effects of reducing conditions on the folding of the spike (S) protein and on the intracellular transport of the membrane (M) protein of the mouse hepatitis coronavirus. These proteins differ in their potential to form disulfide bonds in the lumen of the endoplasmic reticulum (ER). Intrachain disulfide bonds are formed in the S protein but not in M, which was demonstrated in a pulse-chase experiment by analyzing the viral proteins under nonreducing conditions. To reduce disulfide bonds in vivo, we added dithiothreitol (DTT) to the culture medium of mouse hepatitis coronavirus-infected cells following a procedure recently described by Braakman et al. (I. Braakman, J. Helenius, and A. Helenius, EMBO J. 11:1717-1722, 1992). Short exposure to DTT resulted in the complete reduction of newly synthesized S protein and affected its conformation as judged by the change in mobility in nonreducing gels and by the loss of recognition by a conformation-specific monoclonal antibody. Using this antibody in an immunofluorescence assay, we monitored the reducing effect of DTT in situ. DTT was found to initially affect only the S protein present in the ER; also, after longer treatment, the remaining signal also gradually disappeared. In contrast, folding and transport of the M protein were not inhibited by DTT. Under reducing conditions, M was transported efficiently to the trans side of the Golgi complex, indicating that cellular processes such as ER-to-Golgi transport, O-glycosylation, and Golgi retention were unaffected. In the presence of DTT, the M protein even moved at an increased rate to the Golgi complex, which is probably because of its failure to interact with unfolded S protein. The effects of in vivo reduction were reversible. When DTT was removed from pulse-labeled cells, the S protein folded posttranslationally and aberrantly; during its oxidation, most of S now transiently aggregated into large disulfide-linked complexes from which subsequently folded S molecules dissociated.     

Pathways for protein disulphide bond formation

The folding of many secretory proteins depends upon the formation of disulphide bonds. Recent advances in genetics and cell biology have outlined a core pathway for disulphide bond formation in the endoplasmic reticulum (ER) of eukaryotic cells. In this pathway, oxidizing equivalents flow from the recently identified ER membrane protein Ero1p to secretory proteins via protein disulphide isomerase (PDI). Contrary to prior expectations, oxidation of glutathione in the ER competes with oxidation of protein thiols. Contributions of PDI homologues to the catalysis of oxidative folding will be discussed, as will similarities between eukaryotic and prokaryotic disulphide-bond-forming systems.     

The endoplasmic reticulum and the unfolded protein response

The endoplasmic reticulum (ER) is the site where proteins enter the secretory pathway. Proteins are translocated into the ER lumen in an unfolded state and require protein chaperones and catalysts of protein folding to attain their final appropriate conformation. A sensitive surveillance mechanism exists to prevent misfolded proteins from transiting the secretory pathway and ensures that persistently misfolded proteins are directed towards a degradative pathway. In addition, those processes that prevent accumulation of unfolded proteins in the ER lumen are highly regulated by an intracellular signaling pathway known as the unfolded protein response (UPR). The UPR provides a mechanism by which cells can rapidly adapt to alterations in client protein-folding load in the ER lumen by expanding the capacity for protein folding. In addition, a variety of insults that disrupt protein folding in the ER lumen also activate the UPR. These include changes in intralumenal calcium, altered glycosylation, nutrient deprivation, pathogen infection, expression of folding-defective proteins, and changes in redox status. Persistent protein misfolding initiates apoptotic cascades that are now known to play fundamental roles in the pathogenesis of multiple human diseases including diabetes, atherosclerosis and neurodegenerative diseases.     

Posttranslational folding of vesicular stomatitis virus G protein in the ER: involvement of noncovalent and covalent complexes

In this study, we show that posttranslational folding of Vesicular Stomatitis virus G protein subunits can involve noncovalent, multimeric complexes as transient intermediates. The complexes are heterogeneous in size (4-21S20,W), contain several G glycopolypeptides, and are associated with BiP/GRP78. The newly synthesized, partially intrachain disulfide-bonded G proteins enter these complexes immediately after chain termination, and are released 1-4 min later as fully oxidized, trimerization-competent monomers. These monomers are properly folded, judging by their binding of conformation-specific mAbs. When the G protein is translated in the presence of DTT, it remains reduced, largely unfolded and aggregated in the ER, but it can fold successfully when the DTT is removed. In this case, contrary to normal folding, the aggregates become transiently disulfide cross-linked. We also demonstrated that the fidelity of the folding process is dependent on metabolic energy. Finally, we established that the G protein of the folding mutant of the Vesicular Stomatitis virus, ts045, is blocked at a relatively late step in the folding pathway and remains associated with oligomeric, BiP/GRP78-containing folding complexes.     

The manganese cation disrupts membrane dynamics along the secretory pathway

The endoplasmic reticulum and Golgi apparatus play key roles in regulating the folding, assembly, and transport of newly synthesized proteins along the secretory pathway. We find that the divalent cation manganese disrupts the Golgi apparatus and endoplasmic reticulum (ER). The Golgi apparatus is fragmented into smaller dispersed structures upon manganese treatment. Golgi residents, such as TGN46, beta1,4-galactosyltransferase, giantin, and GM130, are still segregated and partitioned correctly into smaller stacked fragments in manganese-treated cells. The mesh-like ER network is substantially affected and peripheral ER elements are collapsed. These effects are consistent with manganese-mediated inhibition of motor proteins that link membrane organelles along the secretory pathway to the cytoskeleton. This divalent cation thus represents a new tool for studying protein secretion and membrane dynamics along the secretory pathway.     

Biosynthetic protein transport through the early secretory pathway

 Newly synthesized proteins destined for delivery to the cell surface are inserted cotranslationally into the endoplasmic reticulum (ER) and, after their correct folding, are transported out of the ER. During their transport to the cell surface, cargo proteins pass through the various cisternae of the Golgi apparatus and, in the trans-most cisternae of the stack, are sorted into constitutive secretory vesicles that fuse with the plasma membrane. Simultaneously with anterograde protein transport, retrograde protein transport occurs within the Golgi complex as well as from the Golgi back to the ER. Vesicular transport within the early secretory pathway is mediated by two types of non-clathrin coated vesicles: COPI- and COPII-coated vesicles. The formation of these carrier vesicles depends on the recruitment of cytosolic coat proteins that are thought to act as a mechanical device to shape a flattened donor membrane into a spherical vesicle. A general molecular machinery that mediates targeting and fusion of carrier vesicles has been identified as well. Beside a general overview of the various coat structures known today, we will discuss issues specifically related to the biogenesis of COPI-coated vesicles: (1) a possible role of phospholipase D in the formation of COPI-coated vesicles; (2) a functional role of a novel family of transmembrane proteins, the p24 family, in the initiation of COPI assembly; and (3) the direction COPI-coated vesicles may take within the early secretory pathway. Moreover, we will consider two alternative mechanisms of protein transport through the Golgi stack: vesicular transport versus cisternal maturation. Accepted: 24 October 1997     

Protein-specific features of the general secretion pathway in yeast: the secretion of acid phosphatase

The major phosphate-repressible acid phosphatase (APase) of Saccharomyces cerevisiae, a cell wall glycoprotein, has been extensively used as a reporter protein to analyse successive steps in the yeast secretory pathway. In contrast to other yeast secretory proteins, APase can still be translocated into the endoplasmic reticulum (ER) even when it is made without its signal peptide. This property illustrates the permissiveness of targeting to the ER in yeast. Studies on APase-containing hybrid proteins have provided some of the evidence that specific soluble factors must interact with secretory proteins prior to their translocation across the ER membrane. A systematic analysis of mutations affecting the sequence of the APase signal peptide cleavage site demonstrated that cleavage occurs only when the last amino acid of the signal sequence is small and neutral. This was one of the first studies to verify the requirements for signal peptidase cleavage that had previously only been predicted from statistical analysis. Studies performed either with inhibitors of glycosylation or with mutant APases demonstrated the critical role of core glycosylation for APase folding, which is essential for efficient transport beyond the ER. Following the fate of particular modified APases along the secretory pathway provided insights into some general properties of the secretory apparatus and illustrated the specific requirements for a given protein during its intracellular traffic.     

Protein retention and localization in the endoplasmic reticulum and the golgi apparatus.

Protein transport along the secretory pathway is supported by a noria of vesicles that bud and fuse, load and unload their cargo from one compartment into the other. However, despite this constant flow-through of proteins and lipids the various compartments of the secretory pathway are able to maintain their own specific composition. Here, we discuss recent insights into mechanisms of protein retention and localization that are necessary for the maintenance of endoplasmic reticulum (ER)- and Golgi-associated typical functions such as protein folding and glycosylation in plant cells.     

For whom the bell tolls: protein quality control of the endoplasmic reticulum and the ubiquitin-proteasome connection

The surveillance of the structural fidelity of the proteome is of utmost importance to all cells. The endoplasmic reticulum (ER) is the organelle responsible for proper folding and delivery of proteins to the secretory pathway. It contains a sophisticated protein proofreading and elimination mechanism. Failure of this machinery leads to disease and, finally, to cell death. Elimination of misfolded proteins requires retrograde transport across the ER membrane and depends on the central cytoplasmic proteolytic machinery involved in cellular regulation: the ubiquitin-proteasome system. The basics of this process as well as recent advances in the field are reviewed.     

ERG30, a VAP-33-related protein, functions in protein transport mediated by COPI vesicles.

Intracellular transport of newly synthesized and mature proteins via vesicles is controlled by a large group of proteins. Here we describe a ubiquitous rat protein-endoplasmic reticulum (ER) and Golgi 30-kD protein (ERG30)-which shares structural characteristics with VAP-33, a 33-kD protein from Aplysia californica which was shown to interact with the synaptic protein VAMP. The transmembrane topology of the 30-kD ERG30 corresponds to a type II integral membrane protein, whose cytoplasmic NH(2) terminus contains a predicted coiled-coil motif. We localized ERG30 to the ER and to pre-Golgi intermediates by biochemical and immunocytochemical methods. Consistent with a role in vesicular transport, anti-ERG30 antibodies specifically inhibit intra-Golgi transport in vitro, leading to significant accumulation of COPI-coated vesicles. It appears that ERG30 functions early in the secretory pathway, probably within the Golgi and between the Golgi and the ER.     

Folding and oligomerization of influenza hemagglutinin in the ER and the intermediate compartment.

Influenza hemagglutinin (HA) was used to analyze the stepwise folding and oligomeric assembly of glycoproteins in the early secretory pathway of living cells. In addition to mature trimers, six distinct maturation intermediates were identified. Of these, all the incompletely oxidized forms were located in the endoplasmic reticulum (ER) and associated with calnexin, a membrane-bound, lectin-like ER chaperone. Once fully oxidized, the HA dissociated from calnexin as a monomer, which rapidly became resistant to dithiothreitol (DTT) reduction. Part of these extensively folded molecules moved as monomers into the intermediate compartment between the ER and the Golgi complex. Assembly of homotrimers occurred without calnexin-involvement within the ER and in the intermediate compartment. When anchor-free HA molecules were analyzed, it was found that they reach the DTT-resistant monomeric conformation but fail to trimerize. Taken together, the results provide a definition and intracellular localization of several intermediates in the conformational maturation of HA, including the immediate precursor for trimer assembly.     

Activation of mammalian unfolded protein response is compatible with the quality control system operating in the endoplasmic reticulum

Newly synthesized secretory and transmembrane proteins are folded and assembled in the endoplasmic reticulum (ER) where an efficient quality control system operates so that only correctly folded molecules are allowed to move along the secretory pathway. The productive folding process in the ER has been thought to be supported by the unfolded protein response (UPR), which is activated by the accumulation of unfolded proteins in the ER. However, a dilemma has emerged; activation of ATF6, a key regulator of mammalian UPR, requires intracellular transport from the ER to the Golgi apparatus. This suggests that unfolded proteins might be leaked from the ER together with ATF6 in response to ER stress, exhibiting proteotoxicity in the secretory pathway. We show here that ATF6 and correctly folded proteins are transported to the Golgi apparatus via the same route and by the same mechanism under conditions of ER stress, whereas unfolded proteins are retained in the ER. Thus, activation of the UPR is compatible with the quality control in the ER and the ER possesses a remarkable ability to select proteins to be transported in mammalian cells in marked contrast to yeast cells, which actively utilize intracellular traffic to deal with unfolded proteins accumulated in the ER.     

GRP94, an ER chaperone with protein and peptide binding properties

GRP94 is the ER representative of the HSP90 family of stress-induced proteins. It binds to a limited number of proteins in the secretory pathway, apparently by recognizing advanced folding intermediates or incompletely assembled proteins, GRP94 also binds peptides and can act as a tumor vaccine, delivering the peptides for presentation to T lymphocytes. Here, we review the current data about GRP94 and propose a structural model that integrates the biochemical data and known functions of the protein.     

ER stress and the unfolded protein response

Conformational diseases are caused by mutations altering the folding pathway or final conformation of a protein. Many conformational diseases are caused by mutations in secretory proteins and reach from metabolic diseases, e.g. diabetes, to developmental and neurological diseases, e.g. Alzheimer's disease. Expression of mutant proteins disrupts protein folding in the endoplasmic reticulum (ER), causes ER stress, and activates a signaling network called the unfolded protein response (UPR). The UPR increases the biosynthetic capacity of the secretory pathway through upregulation of ER chaperone and foldase expression. In addition, the UPR decreases the biosynthetic burden of the secretory pathway by downregulating expression of genes encoding secreted proteins. Here we review our current understanding of how an unfolded protein signal is generated, sensed, transmitted across the ER membrane, and how downstream events in this stress response are regulated. We propose a model in which the activity of UPR signaling pathways reflects the biosynthetic activity of the ER. We summarize data that shows that this information is integrated into control of cellular events, which were previously not considered to be under control of ER signaling pathways, e.g. execution of differentiation and starvation programs.