Intracellular processing and transport of NH2-terminally truncated forms of a hemagglutinin-neuraminidase type II glycoprotein

Six amino-terminal deletion mutants of the NH2-terminally anchored (type II orientation) hemagglutinin-neuraminidase (HN) protein of parainfluenza virus type 3 were expressed in tissue culture by recombinant SV-40 viruses. The mutations consisted of progressive deletions of the cytoplasmic domain and, in some cases, of the hydrophobic signal/anchor. Three activities were dissociated for the signal/anchor: membrane insertion, translocation, and anchoring/transport. HN protein lacking the entire cytoplasmic tail was inserted efficiently into the membrane of the endoplasmic reticulum but was translocated inefficiently into the lumen. However, the small amounts that were successfully translocated appeared to be processed subsequently in a manner indistinguishable from that of parental HN. Thus, the cytoplasmic domain was not required for maturation of this type II glycoprotein. Progressive deletions into the membrane anchor restored efficient translocation, indicating that the NH2-terminal 44 amino acids were fully dispensable for membrane insertion and translocation and that a 10-amino acid hydrophobic signal sequence was sufficient for both activities. These latter HN molecules appeared to be folded authentically as assayed by hemagglutination activity, reactivity with a conformation-specific antiserum, correct formation of intramolecular disulfide bonds, and homooligomerization. However, most (85-90%) of these molecules accumulated in the ER. This showed that folding and oligomerization into a biologically active form, which presumably represents a virion spike, occurs essentially to completion within that compartment but is not sufficient for efficient transport through the exocytotic pathway. Protein transport also appeared to depend on the structure of the membrane anchor. These latter mutants were not stably integrated in the membrane, and the small proportion (10-15%) that was processed through the exocytotic pathway was secreted. The maturation steps and some of the effects of mutations described here for a type II glycoprotein resemble previous observations for prototypic type I glycoproteins and are indicative of close similarities in these processes for proteins of both membrane orientations.     

Targeting of membrane proteins to endosomes and lysosomes

The pathways involved in targeting membrane proteins to lysosomes are extraordinarily complex. Newly synthesized proteins in the ER are transported to the Golgi complex, and upon arrival at the trans Golgi network (TGN) are targeted either directly to endosomes, or first to the cell surface from where they can be rapidly internalized into the endocytic pathway for delivery to lysosomes. The routes to endosomes are specified by sorting motifs in the cytoplasmic tails of the proteins that are recognized at the TGN or plasma membrane. The molecular details of these processes are just emerging.     

Endoplasmic reticulum composition and form: Proteins in and out

The endoplasmic reticulum (ER) is the main harbor for newly synthesized proteins in eukaryotic cells. Through a continuous membrane network of sheets and tubules, the ER hosts secretory proteins, integral membrane proteins, and luminal proteins of the endomembrane system. These proteins are translated by ribosomes outside the ER and require subsequent integration into or translocation across the lipid bilayer of the ER. They are then modified post-translationally and folded in the ER. Some of these proteins are packaged into coat protein complex II–coated vesicles for export. Here, we review recent advances in understanding the mechanism of protein translocation and transmembrane domain insertion in the ER, summarize new insights into selective cargo packaging, and discuss the roles of ER morphological dynamics in these processes.     

The making and breaking of the endoplasmic reticulum

The endoplasmic reticulum (ER) is a dynamic organelle central to many essential cellular functions. It is an important calcium store, which functions in cellular signal transduction cascades. It is also the site of entry for secreted proteins into the secretory pathway. Lumenal enzymes will fold and glycosylate these proteins, and if a protein is destined to be secreted, it will be packaged into membrane vesicles that bud off from the ER. The ER is also the site where most cellular lipids are synthesized. It is contiguous with the nuclear envelope, which serves as a diffusion barrier to control entry into and out of the nucleus. In the life cycle of a cell, the ER is in a constant flux of membrane traffic. What maintains the ER in the shape of an intact reticulum among this constant flux of material? We discuss the mechanisms that contribute to the biogenesis of the ER, the maintenance of the organelle, as well as processes that give the ER its characteristic shape and pattern of inheritance.     

Biosynthesis and secretion of pituitary hormones: dynamics and regulation

Production and secretion of hormones by the pituitary involve highly orchestrated intracellular transport and sorting steps. Hormone precursors are routed through a series of compartments before being packaged in secretory granules. These highly dynamic carriers play crucial roles in both prohormone processing and peptide exocytosis. We have employed the ACTH-secreting AtT-20 cell line to study the membrane sorting events that confer functionality (prohormone activation and regulated exocytosis) to these secretory carriers. The unique ability of granules to promote prohormone processing is attributed to their acidic interior. Using a novel avidin-targeted fluorescence ratio imaging technique, we have found that the trans-Golgi of live AtT-20 cells maintains a mildly acidic (approximately pH 6.2) interior. Budding of secretory granules causes the lumen to acidify to 相似文献   

Efficient export of the vesicular stomatitis virus G protein from the endoplasmic reticulum requires a signal in the cytoplasmic tail that includes both tyrosine-based and di-acidic motifs

The vesicular stomatitis virus (VSV) G protein is a model transmembrane glycoprotein that has been extensively used to study the exocytotic pathway. A signal in the cytoplasmic tail of VSV G (DxE or Asp-x-Glu, where x is any amino acid) was recently proposed to mediate efficient export of the protein from the endoplasmic reticulum (ER). In this study, we show that the DxE motif only partially accounts for efficient ER exit of VSV G. We have identified a six-amino-acid signal, which includes the previously identified Asp and Glu residues, that is required for efficient exit of VSV G from the ER. This six-residue signal also includes the targeting sequence YxxO (where x is any amino acid and O is a bulky, hydrophobic residue) implicated in several different sorting pathways. The only defect in VSV G proteins with mutations in the six-residue signal is slow exit from the ER; folding and oligomerization in the ER are normal, and the mutants eventually reach the plasma membrane. Addition of this six-residue motif to an inefficiently transported reporter protein is sufficient to confer an enhanced ER export rate. The signal we have identified is highly conserved among divergent VSV G proteins, and we suggest this reflects the importance of this motif in the evolution of VSV G as a proficient exocytic protein.     

Transport and assembly processes in the endoplasmic reticulum

Until recently, the endoplasmic reticulum (ER) of eukaryotic cells was regarded as an open corridor for the unregulated movement of newly-synthesized exocytotic proteins from their site of membrane translocation to the vesicles that ferry them from the transitional elements of the ER to the Golgi apparatus. Moreover, it was widely assumed that the folding and assembly of newly translocated polypeptides into their tertiary and quaternary structure is a spontaneous process that does not involve the intervention of other cellular proteins. In this article we review evidence that the ER is a highly discriminatory organelle that grants passage only to proteins that have attained an essentially native conformation, and summarize current knowledge about resident ER proteins that appear to facilitate and/or monitor protein folding and assembly in this organelle.     

Intracellular assembly and secretion of recombinant subviral particles from tick-borne encephalitis virus

It is believed that flavivirus assembly occurs by intracellular budding of the nucleocapsid into the lumen of the endoplasmic reticulum (ER). Recombinant expression of tick-borne encephalitis (TBE) virus envelope proteins prM and E in mammalian cells leads to their incorporation into enveloped recombinant subviral particles (RSPs), which have been used as a model system for studying assembly and entry processes and are also promising vaccine candidates. In this study, we analyzed the formation and secretion of TBE virus RSPs and of a membrane anchor-free E homodimer in mammalian cells. Immunofluorescence microscopy showed that E was accumulated in the lumen of the ER. RSPs were observed by electron microscopy in the rough and smooth ER and in downstream compartments of the secretory pathway. About 75% of the particles appeared to be of the size expected for RSPs (about 30 nm in diameter), but a number of larger particles and tubular structures were also observed in these compartments. Secretion of membrane anchor-free E dimers was detected 30 min after synthesis of prM and E, and secretion of RSPs was detected 1 h after synthesis of prM and E. We also found that the presence of the single N-linked oligosaccharide side chain on the E protein and its trimming by glucosidases was necessary for secretion of RSPs and truncated E dimers. Our results suggest that incorporation of prM and E into RSPs occurs at the ER membrane without other viral elements being required, followed by rapid transport along the compartments of the secretory pathway and secretion. Moreover, the carbohydrate side chain of E is involved in at least one assembly or transport step.     

A subclass of proteins and sulfated macromolecules secreted by AtT-20 (mouse pituitary tumor) cells is sorted with adrenocorticotropin into dense secretory granules

The AtT-20 cell, a mouse pituitary tumor line that secretes adrenocorticotropin and beta-endorphin, sorts the proteins it externalizes into two exocytotic pathways. Cells that are labeled with [35S]methionine or [35S]sulfate can be shown to transport three acidic polypeptides (65,000, 60,000, and 37,000 mol wt) and at least two sulfated macromolecules into storage secretory granules. When the cells are stimulated by the secretagogue 8-bromo-cAMP, these polypeptides are coordinately secreted with mature adrenocorticotropin into the culture medium. In contrast, a completely different set of secreted polypeptides and sulfated macromolecules does not enter a storage form and is transported to the cell surface more rapidly. Their secretion from the cells is constitutive and does not require the presence of secretagogues. These molecules, like a viral membrane glycoprotein described previously (Gumbiner, B., and R. B. Kelly, 1982, Cell, 28:51-59) are not found in isolated secretory granules and therefore must reach the cell surface in a different exocytotic vesicle. The segregation of a subclass of secretory macromolecules into the secretory granules, despite the existence of another potential secretory pathway, suggests that these molecules have specific functions related to regulated hormone secretion or storage. Presumably all of the proteins secreted by the regulated secretory granule pathway share some common property that targets them to the secretory granule.     

Regulation of exocytosis in adrenal chromaffin cells: focus on ARF and Rho GTPases

Neurons and neuroendocrine cells release transmitters and hormones by exocytosis, a highly regulated process in which secretory vesicles or granules fuse with the plasma membrane to release their contents in response to a calcium trigger. Several stages have been recognized in exocytosis. After recruitment and docking at the plasma membrane, vesicles/granules enter a priming step, which is then followed by the fusion process. Cortical actin remodelling accompanies the exocytotic reaction, but the links between actin dynamics and trafficking events remain poorly understood. Here, we review the action of Rho and ADP-ribosylation factor (ARF) GTPases within the exocytotic pathway in adrenal chromaffin cells. Rho proteins are well known for their pivotal role in regulating the actin cytoskeleton. ARFs were originally identified as regulators of vesicle transport within cells. The possible interplay between these two families of GTPases and their downstream effectors provides novel insights into the mechanisms that govern exocytosis.     

The transmembrane domain of the respiratory syncytial virus F protein is an orientation-independent apical plasma membrane sorting sequence

The processes that facilitate transport of integral membrane proteins though the secretory pathway and subsequently target them to particular cellular membranes are relevant to almost every field of biology. These transport processes involve integration of proteins into the membrane of the endoplasmic reticulum (ER), passage from the ER to the Golgi, and post-Golgi trafficking. The respiratory syncytial virus (RSV) fusion (F) protein is a type I integral membrane protein that is uniformly distributed on the surface of infected nonpolarized cells and localizes to the apical plasma membrane of polarized epithelial cells. We expressed wild-type or altered RSV F proteins to gain a better understanding of secretory transport and plasma membrane targeting of type I membrane proteins in polarized and nonpolarized epithelial cells. Our findings reveal a novel, orientation-independent apical plasma membrane targeting function for the transmembrane domain of the RSV F protein in polarized epithelial cells. This work provides a basis for a more complete understanding of the role of the transmembrane domain and cytoplasmic tail of viral type I integral membrane proteins in secretory transport and plasma membrane targeting in polarized and nonpolarized cells.     

A non-autophagic pathway for diversion of ER secretory proteins to lysosomes

Intracisternal granules (ICG) develop in the rough ER of hyperstimulated thyrotrophs or thyroid hormone-secreting cells of the anterior pituitary gland. To determine the fate of these granules, we carried out morphological and immunocytochemical studies on pituitaries of thyroxine-treated, thyroidectomized rats. Under these conditions the ER of thyrotrophs is dramatically dilated and contains abundant ICG; the latter contain beta subunits of thyrotrophic hormone (TSH-beta). Based on purely morphologic criteria, intermediates were identified that appeared to represent stages in the transformation of a part rough/part smooth ER cisterna into a lysosome. Using immunocytochemical and cytochemical markers, two major types of intermediates were distinguished: type 1 lacked ribosomes but were labeled with antibodies against both ER markers (PDI, KDEL, ER membrane proteins) and a lysosomal membrane marker, lgp120. They also were reactive for the lysosomal enzyme, acid phosphatase, by enzyme cytochemistry. Type 2 intermediates were weakly reactive for ER markers and contained both lgp120 and lysosomal enzymes (cathepsin D, acid phosphatase). Taken together these results suggest that in hyperstimulated thyrotrophs part rough/part smooth ER elements containing ICG lose their ribosomes, their membrane is modified, and they sequentially acquire a lysosome-type membrane and lysosomal enzymes. The findings are compatible with the conclusion that a pathway exists by which under certain conditions, secretory proteins present in the ER as well as ER membrane and content proteins can be degraded by direct conversion of ER cisternae into lysosomes.     

An N-terminal double-arginine motif maintains type II membrane proteins in the endoplasmic reticulum.

Use of alternative initiator methionines in human invariant (Ii) chain mRNA results in the synthesis of two polypeptides, Iip33 and Iip31. After synthesis both isoforms are inserted into the endoplasmic reticulum (ER) as type II membrane proteins. Subsequently, Iip31 is transported out of the ER, guiding MHC class II to the endocytic pathway, whereas Iip33, which differs by only a 16 residue extension at the N-terminus, becomes an ER resident. Mutagenesis of this extension showed that multiple arginines close to the N-terminus were responsible for ER targeting. The minimal requirements of this targeting motif were found to be two arginines (RR) located at positions 2 and 3, 3 and 4 or 4 and 5 or split by a residue at positions 2 and 4 or 3 and 5. Transplanting an RR motif onto transferrin receptor demonstrated that this motif can target other type II membrane proteins to the ER. The characteristics of this RR motif are similar to the KK ER targeting motif for type I membrane proteins. Indeed, RR-tagged transferrin receptor partially localized to the intermediate compartment, suggesting that like the KK motif, the RR motif directs the retrieval of membrane proteins to the ER via a retrograde transport pathway.     

Assembly-dependent trafficking assays in the detection of receptor-receptor interactions

Assembly-dependent trafficking is a property of many multimeric membrane protein complexes; this coupling of assembly and trafficking processes provides an important cellular quality control mechanism, ensuring that only properly folded and assembled complexes are expressed on the cell surface. In all membrane protein complexes whose trafficking is known to be assembly-dependent, at least one of the subunits contains an endoplasmic reticulum (ER) retention/retrieval signal that is shielded on subunit assembly, allowing the assembled protein complex to traffic to the plasma membrane. Under these conditions, presence of the normally retained subunit on the cell surface can be used as an indirect index of protein assembly in the ER. In this article, I describe the design of two complementary approaches (trafficking enhancement and trap assays) that can be used separately or in combination to determine whether two (or more) proteins assemble in the ER, i.e., whether they constitutively oligomerize. Both of the approaches are based on the measurement of plasma membrane-expressed proteins using antibody-mediated detection of extracellularly expressed epitopes and subsequent luminometric quantification. These methods provide a straightforward and relatively inexpensive way to assess protein-protein interactions early in the synthetic pathway.     

Molecular basis for sculpting the endoplasmic reticulum membrane

The endoplasmic reticulum (ER) is involved in many critical processes, including protein and lipid synthesis and calcium storage. Morphologically, the ER can be divided into two subdomains: a network of interconnected tubules and interspersed sheets. Until recently, how these different compartments form in a continuous membrane system was unclear. Several classes of integral membrane proteins have been identified in the ER; the reticulons and DP1/Yop1p play roles in the generation of ER tubules, and possibly in stabilizing ER sheets, atlastins and Sey1p are dynamin-like GTPases that facilitate tubular network formation by mediating ER membrane fusion, and Climp63, p180, and kinectin are enriched in ER sheets and influence their formation. In this review, we summarize recent advances in our understanding of how these proteins participate in ER shaping. We also discuss possible mechanisms for regulating ER morphology via the cytoskeleton. Lessons learned about sculpting the ER membrane may be applicable to other organelles.     

Targeting and membrane-insertion of a sunflower oleosin in vitro and in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>: the central hydrophobic domain contains more than one signal sequence,and directs oleosin insertion into the endoplasmic reticulum membrane using a signal anchor sequence mechanism

A range of N- and C-terminal deletions of an oleosin from Helianthus annuus L. were used to study the endoplasmic reticulum (ER) targeting and membrane insertion of this protein both in vitro and in vivo in yeast ( Saccharomyces cerevisiae). Neither the N- nor the C-terminal hydrophilic domains are important for targeting and/or membrane insertion, with all the information required for these processes located within the central hydrophobic region of the protein. However, in vitro membrane-insertion experiments suggest that these domains are important for a correct topology of the oleosin within the ER membrane. The first half of the hydrophobic central domain, flanked by the positively charged N-terminal domain, is likely to function as a type-II signal-anchor (SAII) sequence. However, in the absence of the N-terminal 26 residues of this domain, the proline-knot region and the second half of this hydrophobic domain are sufficient to direct oleosin to the ER and to allow stable (but far less efficient) integration of the protein into the membrane. Taken together, these results indicate that oleosin contains more than one domain that is capable of interacting with the signal recognition particle to direct the protein to the ER membrane.     

Multiple molecular determinants in the carboxyl terminus regulate dopamine transporter export from endoplasmic reticulum

The plasma membrane dopamine transporter (DAT) has an essential role in terminating dopaminergic neurotransmission by reuptake of dopamine into the presynaptic neurons. Therefore, the amount of DAT at the cell surface is a critical determinant of DAT function. In this study, we examined the role of the carboxyl terminus of DAT in trafficking of the transporter through the biosynthetic pathway to the plasma membrane. Live cell fluorescence microscopy and cell surface biotinylation were used to study the effects of systematic deletions and alanine substitutions in the carboxyl terminus on DAT localization. It was found that alanine substitutions of Lys-590 and Asp-600 significantly delayed the delivery of DAT to the plasma membrane because of retention of DAT in the endoplasmic reticulum (ER). Most surprising, mutation of Gly-585 to alanine completely blocked the exit of DAT from the ER and surface expression of the transporter. The effect of these three mutations on ER export of DAT was demonstrated in porcine aortic endothelial cells and the immortalized neuronal cell line 1RB3AN27. In primary cultures of rat embryonic midbrain neurons, DAT G585A, K590A, and D600A mutants were restricted to the cell soma and did not traffic to the dendrites or axonal processes. These data are consistent with the model whereby the local conformation and/or intramolecular interactions of the sequences of the DAT carboxyl terminus proximal to the last transmembrane domain are essential for the ER export of the transporter.     

The early stages of the intracellular transport of membrane proteins: clinical and pharmacological implications

Intracellular transport mechanisms ensure that integral membrane proteins are delivered to their correct subcellular compartments. Efficient intracellular transport is a prerequisite for the establishment of both cell architecture and function. In the past decade, transport processes of proteins have also drawn the attention of clinicians and pharmacologists since many diseases have been shown to be caused by transport-deficient proteins. Membrane proteins residing within the plasma membrane are transported via the secretory (exocytotic) pathway. The general transport routes of the secretory pathway are well established. The transport of membrane proteins starts with their integration into the ER membrane. The ribosomes synthesizing membrane proteins are targeted to the ER membrane, and the nascent chains are co-translationally integrated into the bilayer, i.e., they are inserted while their synthesis is in progress. During ER insertion, the orientation (topology) of the proteins in the membrane is determined. Proteins are folded, and their folding state is checked by a quality control system that allows only correctly folded forms to leave the ER. Misfolded or incompletely folded forms are retained, transported back to the cytosol and finally subjected to proteolysis. Correctly folded proteins are transported in the membranes of vesicles through the ER/Golgi intermediate compartment (ERGIC) and the individual compartments of the Golgi apparatus (cis, medial, trans) to the plasma membrane. In this review, the current knowledge of the first stages of the intracellular trafficking of membrane proteins will be summarized. This early secretory pathway includes the processes of ER insertion, topology determination, folding, quality control and the transport to the Golgi apparatus. Mutations in the genes of membrane proteins frequently lead to misfolded forms that are recognized and retained by the quality control system. Such mutations may cause inherited diseases like cystic fibrosis or retinitis pigmentosa. In the second part of this review, the clinical implications of the early secretory pathway will be discussed. Finally, new pharmacological strategies to rescue misfolded and transport-defective membrane proteins will be outlined.Abbreviations AP1 Clathrin-associated adaptor protein complex 1 - AQP Aquaporin - ARF ADP-ribosylation factor - AVP 8-Arginine-vasopressin;BiP immunoglobulin heavy chain binding protein - CFTR Cystic fibrosis transmembrane conductance regulator - CLQTS Congenital long QT syndrome - CMT Charcot-Marie-Tooth syndrome - CNX Calnexin - COPI Coat protein complex I - COPII Coat protein complex II - CPX 8-Cyclopentyl-1,2-dipropylxanthine - CRT Calreticulin - CSID Congenital sucrose-isomaltase deficiency - Cx Connexin - cGMP Cyclic 3:5 guanosine monophosphate - ECL Extracellular loop - EndoH Endoglycosidase H - ER Endoplasmic reticulum - ERAD ER-associated degradation - ERGIC ER/Golgi intermediate compartment - ERp ER protein - ET_BR Human endothelin B receptor - FH Familial hypercholesterolemia - GABA Gamma amino butyric acid - GFP Green fluorescent protein - GH Growth hormone - GHIS Growth hormone insensitivity syndrome - GLCase Glucosidase - GlcNac N-acetylglucosamine - GPCR G protein-coupled receptor - GPI Glycosylphosphatidylinositol - G protein GTP-binding protein - GRP Glucose-regulated protein - HA Hemagglutinin - Hdj-2 Human DnaJ-2 protein - HFE Human hemochromatosis protein - HH Hereditary hemochromatosis - HEK 293 cells Human embryonic kidney 293 cells - HERG Human ether-a-go-go-related protein - Hsc70 Heat shock cognate 70 protein - ICL Intracellular loop - IGF-I Insulin-like growth factor-1 - I_Kr Rapidly activating delayed rectifier potassium current - I_Ks Slowly activating delayed rectifier potassium current - JAK Janus kinase - LDL Low-density lipoprotein - LH Luteinizing hormone/choriogonadotropin - LS Laron syndrome - MATP Membrane associated transporter protein - MDCK cells Madin-Darby canine kidney epithelial cells - MHC Major histocompatibility complex - MiRP1 minK-related peptide 1 - NDI Congenital nephrogenic diabetes insipidus - NMDA N-methyl-d-aspartate - OCA Oculocutaneous albinism - PDI Protein disulfide isomerase - Pgp P-glycoprotein - PKA Protein kinase A - PLP Proteolipid protein - PMP22 Peripheral myelin protein 22 - RP Primary retinitis pigmentosa - SI Sucrase-isomaltase - SNARE Ethylmaleimide-sensitive factor attachment protein - SRP Signal recognition particle - TCR T-cell antigen receptor - TM Transmembrane domain - TRAM Translocating chain-associated membrane protein - Tyr Tyrosinase - Tyrp1 Tyrosinase-related protein-1 - UGGT UDP-glucose:glycoprotein glucosyltransferase - VIP Vesicular-integral membrane protein - V₂R Vasopressin V₂ receptor - VSV Vesicular stomatitis virus     

Intracellular transport, assembly, and degradation of wild-type and disease-linked mutant gap junction proteins

More than 130 different mutations in the gap junction integral plasma membrane protein connexin32 (Cx32) have been linked to the human peripheral neuropathy X-linked Charcot-Marie-Tooth disease (CMTX). How these various mutants are processed by the cell and the mechanism(s) by which they cause CMTX are unknown. To address these issues, we have studied the intracellular transport, assembly, and degradation of three CMTX-linked Cx32 mutants stably expressed in PC12 cells. Each mutant had a distinct fate: E208K Cx32 appeared to be retained in the endoplasmic reticulum (ER), whereas both the E186K and R142W mutants were transported to perinuclear compartments from which they trafficked either to lysosomes (R142W Cx32) or back to the ER (E186K Cx32). Despite these differences, each mutant was soluble in nonionic detergent but unable to assemble into homomeric connexons. Degradation of both mutant and wild-type connexins was rapid (t(1/2) < 3 h) and took place at least in part in the ER by a process sensitive to proteasome inhibitors. The mutants studied are therefore unlikely to cause disease by accumulating in degradation-resistant aggregates but instead are efficiently cleared from the cell by quality control processes that prevent abnormal connexin molecules from traversing the secretory pathway.