Vesicle fusion following receptor-mediated endocytosis requires a protein active in Golgi transport

In reconstitution studies N-ethylmaleimide, a sulphydryl alkylating reagent, inhibits both fusion of endocytic vesicles and vesicular transport in the Golgi apparatus. We show here that the same N-ethylmaleimide-sensitive factor that catalyses the vesicle-mediated transport within Golgi stacks is also required for endocytic vesicle fusion. Thus, it is likely that a common mechanism for vesicle fusion exists for both the secretory and endocytic pathways of eukaryotic cells.     

A recycling pathway between the endoplasmic reticulum and the Golgi apparatus for retention of unassembled MHC class I molecules

Assembly of class I major histocompatibility complex (MHC) molecules involves the interaction of two distinct polypeptides (the heavy and light chains) with peptide antigen. Cell lines synthesizing both chains but expressing low levels of MHC class I molecules on their surface as a result of a failure in assembly and transport have been identified. We now report that although the apparent steady-state distribution in these cells of class I molecules is in the endoplasmic reticulum (ER), the molecules in fact are recycled between the ER and Golgi, rather than retained in the ER. This explains the failure of class I molecules to negotiate the secretory pathway. Class I molecules do not seem to be modified by Golgi enzymes, suggesting that the proteins do not reach the Golgi apparatus during recycling. But morphological and subcellular fractionation evidence indicates that they pass through the cis Golgi or a Golgi-associated organelle, which we postulate to be the recycling organelle. This compartment, which we call the 'cis-Golgi network', would thereby be a sorting organelle that selects proteins for return to the ER.     

Quality control in the endoplasmic reticulum protein factory

The endoplasmic reticulum (ER) is a factory where secretory proteins are manufactured, and where stringent quality-control systems ensure that only correctly folded proteins are sent to their final destinations. The changing needs of the ER factory are monitored by integrated signalling pathways that constantly adjust the levels of folding assistants. ER chaperones and signalling molecules are emerging as drug targets in amyloidoses and other protein-conformational diseases.     

Retrograde transport of endocytosed Shiga toxin to the endoplasmic reticulum.

Shiga toxin and some other protein toxins that act on targets in the cytosol have previously been shown to enter the trans-Golgi network. Transport by this route may be necessary for translocation of the toxin to the cytosol and for intoxication, but it is not known whether the enzymatically active part of the toxins actually enters the cytosol from the trans-Golgi network. It has been suggested that such toxins are transported in a retrograde manner to the endoplasmic reticulum and that translocation occurs in this organelle, but retrograde transport of endocytosed material beyond the trans-Golgi network has never been demonstrated. Here we show that in butyric acid-treated A431 cells endocytosed Shiga toxin is not only transported to the trans-Golgi network, but also to all Golgi stacks, to the endoplasmic reticulum and to the nuclear envelope. Furthermore, butyric acid sensitizes the cells to Shiga toxin, which is consistent with the possibility that retrograde transport is required for translocation of the toxin to the cytosol.     

A substrate-specific inhibitor of protein translocation into the endoplasmic reticulum

The segregation of secretory and membrane proteins to the mammalian endoplasmic reticulum is mediated by remarkably diverse signal sequences that have little or no homology with each other. Despite such sequence diversity, these signals are all recognized and interpreted by a highly conserved protein-conducting channel composed of the Sec61 complex. Signal recognition by Sec61 is essential for productive insertion of the nascent polypeptide into the translocation site, channel gating and initiation of transport. Although subtle differences in these steps can be detected between different substrates, it is not known whether they can be exploited to modulate protein translocation selectively. Here we describe cotransin, a small molecule that inhibits protein translocation into the endoplasmic reticulum. Cotransin acts in a signal-sequence-discriminatory manner to prevent the stable insertion of select nascent chains into the Sec61 translocation channel. Thus, the range of substrates accommodated by the channel can be specifically and reversibly modulated by a cell-permeable small molecule that alters the interaction between signal sequences and the Sec61 complex.     

A protein of the endoplasmic reticulum involved early in polypeptide translocation.

To identify components of the mammalian endoplasmic reticulum involved in the translocation of secretory proteins, crosslinking and reconstitution methods were combined. A multispanning abundant membrane glycoprotein was found which is in proximity to nascent chains early in translocation. In reconstituted proteoliposomes, this protein is stimulatory or required for the translocation of secretory proteins.     

Role of ATP and disulphide bonds during protein folding in the endoplasmic reticulum.

Being topologically equivalent to the extracellular space, the lumen of the endoplasmic reticulum (ER) provides a unique folding environment for newly synthesized proteins. Unlike other compartments in the cell where folding occurs, the ER is oxidizing and therefore can promote the formation of disulphide bonds. The reducing agent dithiothreitol, when added to living cells, inhibits disulphide formation with profound effects on folding. Taking advantage of this effect, we demonstrate here that folding of influenza haemagglutinin is energy dependent. Metabolic energy is required to support the correct folding and disulphide bond formation in this well characterized viral glycoprotein, to rescue misfolded proteins from disulphide-linked aggregates, and to maintain the oxidized protein in its folded and oligomerization-competent state.     

Prevention of HIV-1 glycoprotein transport by soluble CD4 retained in the endoplasmic reticulum

The envelope glycoprotein (gp120/41) of the human immunodeficiency virus (HIV-1) attaches the virus to the cellular CD4 receptor and mediates virus entry into the cytoplasm. In addition to being required for formation of infectious HIV, expression of gp120/41 at the plasma membrane causes the cytopathic fusion of cells carrying the CD4 antigen. The expression of gp120/41 is therefore an ideal target for therapeutic strategies designed to combat AIDS. Here we show that expression of a soluble CD4 molecule, mutated to contain a specific retention signal for the endoplasmic reticulum, blocks secretion of gp120 and surface expression of gp120/41, but does not interfere with transport of wild-type CD4. By blocking transport of the HIV glycoprotein, this retained CD4 molecule prevents the fusion of CD4 cells that is normally caused by the HIV glycoprotein. Expression of the retained CD4 molecule in human T cells might therefore be useful in the intracellular immunization procedure suggested by Baltimore.     

A signal sequence receptor in the endoplasmic reticulum membrane

Protein translocation across the endoplasmic reticulum (ER) membrane is triggered at several stages by information contained in the signal sequence. Initially, the signal sequence of a nascent secretory protein upon emergence from the ribosome is recognized by a polypeptide of relative molecular mass 54,000 (Mr54K) which is part of the signal recognition particle (SRP). Binding of SRP may induce a site-specific elongation arrest of translation in vitro. Attachment of the arrested translation complex to the ER membrane is mediated by the SRP-receptor (docking protein) and is accompanied by displacement of the SRP from both the ribosome and the signal sequence. We have investigated the fate of the signal sequence following the disengagement of SRP and its receptor by a crosslinking approach. We report here that the signal sequence of nascent preprolactin, after its release from the SRP, interacts with a newly discovered component, a signal sequence receptor (SSR), which is an integral, glycosylated protein of the rough ER membrane (Mr approximately 35K).     

Signal recognition particle contains a 7S RNA essential for protein translocation across the endoplasmic reticulum

In addition to its previously characterized, six different polypeptide components, signal recognition protein--which functions in protein translocation across and integration into the endoplasmic reticulum membrane--contains a 7S RNA molecule. The RNA is closely identified with the small cytoplasmic 7SL RNA and is required for both structural and functional properties of signal recognition protein--which we therefore rename signal recognition particle.     

Characterization of the rough endoplasmic reticulum ribosome-binding activity.

The rough endoplasmic reticulum membranes of mammalian cells contain specific ribosome-binding sites. A purification to apparent homogeneity of a negatively charged protein (ERp180) of relative molecular mass 180,000 (180 K) was reported which was proposed to function as a rough endoplasmic reticulum ribosome receptor. We report here that ribosome-binding site activity quantitatively solubilized from rough endoplasmic reticulum membranes does not cofractionate with ERp180. By contrast, ribosome-binding site activity fractionates as a much smaller, positively charged protein.     

Recognition of transmembrane helices by the endoplasmic reticulum translocon

Membrane proteins depend on complex translocation machineries for insertion into target membranes. Although it has long been known that an abundance of nonpolar residues in transmembrane helices is the principal criterion for membrane insertion, the specific sequence-coding for transmembrane helices has not been identified. By challenging the endoplasmic reticulum Sec61 translocon with an extensive set of designed polypeptide segments, we have determined the basic features of this code, including a 'biological' hydrophobicity scale. We find that membrane insertion depends strongly on the position of polar residues within transmembrane segments, adding a new dimension to the problem of predicting transmembrane helices from amino acid sequences. Our results indicate that direct protein-lipid interactions are critical during translocon-mediated membrane insertion.     

Protein translocation across the eukaryotic endoplasmic reticulum and bacterial plasma membranes

A decisive step in the biosynthesis of many proteins is their partial or complete translocation across the eukaryotic endoplasmic reticulum membrane or the prokaryotic plasma membrane. Most of these proteins are translocated through a protein-conducting channel that is formed by a conserved, heterotrimeric membrane-protein complex, the Sec61 or SecY complex. Depending on channel binding partners, polypeptides are moved by different mechanisms: the polypeptide chain is transferred directly into the channel by the translating ribosome, a ratcheting mechanism is used by the endoplasmic reticulum chaperone BiP, and a pushing mechanism is used by the bacterial ATPase SecA. Structural, genetic and biochemical data show how the channel opens across the membrane, releases hydrophobic segments of membrane proteins laterally into lipid, and maintains the membrane barrier for small molecules.     

Location of MHC-encoded transporters in the endoplasmic reticulum and cis-Golgi.

Immune recognition of intracellular proteins is mediated by major histocompatibility complex (MHC) class I molecules that present short peptides to cytotoxic T cells. Evidence suggests that peptides arise by cleavage of proteins in the cytoplasm and are transported by a signal-independent mechanism into a pre-Golgi region of the cell, where they take part in the assembly of class I heavy chains with beta 2-microglobulin (reviewed in refs 5-7). Analysis of cells that have defects in class I molecule assembly and antigen presentation has shown that this phenotype can result from mutations in either of the two ABC transporter genes located in the class II region of the MHC. This suggested that the protein complex encoded by these two genes transports peptides from the cytosol into the endoplasmic reticulum. Here we report additional evidence by showing that the transporter complex is located in the endoplasmic reticulum membrane and is probably oriented with its ATP-binding domains in the cytosol.     

A proteome-wide screen identifies valosin-containing protein as an essential regulator of podocyte endoplasmic reticulum stress

To investigate proteins expressed in the renal tissue of the passive Heymann nephritis (pHN) rat model,we prepared pHN rat models with anti-FxA1 serum and analyzed the proteins differentially expressed in the kidney tissue with label-free liquid chromatography-tandem mass spectrometry.We then analyzed in depth the endoplasmic reticulum stress (ERS)-related protein using an online bioinformatics platform.Forty-one differential proteins and their annotations were obtained.Gene Ontology (GO) function analysis showed that 16 proteins were involved in cellular metabolism and 22 were proteins related to catalytic activity,including protein folding or ATPase.Protein-GO networks indicated that VCP could interact with the ERS marker HSPa5,with both involved in a single pathway.On inhibition of podocyte VCP by RNAi under normal conditions,the HSPa5 expression level did not change,but when the cell was subjected to ERS by tunicamycin,HSPa5 expression significantly increased with RNAi of VCP when compared with the tunicamycin-treated group.Our results showed that ERS plays an important role in podocyte injury of membranous nephropathy and is mediated by an HSPa5-VCP signaling pathway,in which the most predominant proteins are those related to cellular metabolism and catalytic activity.     

'Coatomer': a cytosolic protein complex containing subunits of non-clathrin-coated Golgi transport vesicles

Golgi-derived coated vesicles contain a set of coat proteins of relative molecular mass 160,000 (Mr 160K; alpha-COP), 110K (beta-COP), 98K (gamma-COP) and 61K (delta-COP), and several smaller subunits. We have now identified and purified a cytosolic complex containing the same four coat proteins as those of Golgi transport vesicles. We term this complex the Golgi coat promoter or 'coatomer'. The coatomer also contains polypeptides of Mr 36K, 35K and 20K. It represents about 0.2% of soluble cytosolic protein. Gel filtration of unfractionated cytosol indicates that beta-COP resides exclusively in the coatomer complex. The complex seems to be a likely candidate for the unassembled precursor of Golgi coated vesicles, and its purification should help investigations of the role of coat proteins in membrane budding, for which it is necessary to use a refined cell-free system.     

Identification of a ribosome receptor in the rough endoplasmic reticulum

Attachment of ribosomes to the membrane of the endoplasmic reticulum is one of the crucial first steps in the transport and secretion of intracellular proteins in mammalian cells. The process is mediated by an integral membrane protein of relative molecular mass 180,000 (Mr 180K), having a large (at least 160K) cytosolic domain that, when proteolytically detached from the membrane, can competitively inhibit the binding of ribosomes to intact membranes. Isolation of this domain has led to the identification, purification and characterization of the intact ribosome receptor, as well as its functional reconstitution into lipid vesicles.     

AB5 subtilase cytotoxin inactivates the endoplasmic reticulum chaperone BiP

AB5 toxins are produced by pathogenic bacteria and consist of enzymatic A subunits that corrupt essential eukaryotic cell functions, and pentameric B subunits that mediate uptake into the target cell. AB5 toxins include the Shiga, cholera and pertussis toxins and a recently discovered fourth family, subtilase cytotoxin, which is produced by certain Shiga toxigenic strains of Escherichia coli. Here we show that the extreme cytotoxicity of this toxin for eukaryotic cells is due to a specific single-site cleavage of the essential endoplasmic reticulum chaperone BiP/GRP78. The A subunit is a subtilase-like serine protease; structural studies revealed an unusually deep active-site cleft, which accounts for its exquisite substrate specificity. A single amino-acid substitution in the BiP target site prevented cleavage, and co-expression of this resistant protein protected transfected cells against the toxin. BiP is a master regulator of endoplasmic reticulum function, and its cleavage by subtilase cytotoxin represents a previously unknown trigger for cell death.     

Independent phosphatidylinositol synthesis in pituitary plasma membrane and endoplasmic reticulum

Phosphatidylinositol (PtdIns), the most abundant phosphoinositide, is the precursor of phosphatidylinositol 4-monophosphate which is converted to phosphatidylinositol 4,5-bisphosphate, the lipid hydrolysed as an early step in signal transduction by many stimuli. It is generally thought that a single enzyme in the endoplasmic reticulum, PtdIns synthase (CDP-diglyceride:myoinositol 3-phosphatidyltransferase, EC 2.7.8.11), is responsible for PtdIns synthesis and that newly synthesized PtdIns is transported to the plasma membrane by exchange proteins. Several investigators have proposed that there are two functionally distinct pools of PtdIns, one responsive to stimulation and the other not, and that the stimulus-responsive pool may be synthesized at a different site within the cell, perhaps within the plasma membrane. Indeed, it was suggested that there is PtdIns synthase activity in plasma membrane isolated from rat liver. GH3 rat pituitary tumour cells are an excellent model system to study stimulation of phosphoinositide metabolism by thyrotropin-releasing hormone (TRH). Conversion of PtdIns to polyphosphoinositides and TRH (and GTP)-activated phosphoinositide hydrolysis are known to occur in plasma membrane isolated from GH3 cells. Here we report that PtdIns synthase activity in the plasma membrane of GH3 cells is distinct from that present in the endoplasmic reticulum. The plasma membrane PtdIns synthase may be responsible for a portion of PtdIns re-synthesis that occurs during cell stimulation.