Release from Endoplasmic Reticulum Matrix Proteins Controls Cell Surface Transport of MHC Class I Molecules

The anterograde transport of secretory proteins from the endoplasmic reticulum (ER) to the plasma membrane is a multi‐step process. Secretory proteins differ greatly in their transport rates to the cell surface, but the contribution of each individual step to this difference is poorly understood. Transport rates may be determined by protein folding, chaperone association in the ER, access to ER exit sites (ERES) and retrieval from the ER‐Golgi intermediate compartment or the cis‐Golgi to the ER. We have used a combination of folding and trafficking assays to identify the differential step in the cell surface transport of two natural allotypes of the murine major histocompatibility complex (MHC) class I peptide receptor, H‐2D^b and H‐2K^b. We find that a novel pre‐ER exit process that acts on the folded lumenal part of MHC class I molecules and that drastically limits their access to ERES accounts for the transport difference of the two allotypes. Our observations support a model in which the cell surface transport of MHC class I molecules and other type I transmembrane proteins is governed by the affinity of all their folding and maturation states to the proteins of the ER matrix.      

Structural organization of ultrarapidly frozen barley aleurone cells actively involved in protein secretion

The ultrastructural organization of actively secreting barley (Hordeum vulgare L. cv. Himalaya) aleurone cells was examined using ultrarapid-freezing (<-10 000°C s^-1) followed by freeze-fracture and freeze-substitution. Our analysis indicates that much of the evidence supporting a direct pathway from the endoplasmic reticulum (ER) to the plasma membrane (i.e. bypassing the Golgi apparatus) for the secretion of -amylase (EC 3.2.1.1) may not be valid. Cryofixed ER cisternae show no sign of vesiculation during active -amylase secretion in gibberellic acid (GA₃)-treated cells. At the same time, Golgi complexes are abundant and numerous small vesicles are associated with the edges of the cisternae. Vesicles appear to be involved in the delivery of secretory products to the plasma membrane since depressions containing excess membrane material appear there. Treatment with GA₃ also induces changes in the composition of Golgi membranes; most notably, the density of intramembrane particles increases from 2700 m^-2 to 3800 m^-2 because of an increase of particles in the 3–8.5-nm size range. A slight decrease in 9–11-nm particles also occurs. These changes in membrane structure appear to occur as the Golgi complex becomes committed to the processing and packaging of secretory proteins. We suggest that secretory proteins in this tissue are synthesized in the abundant rough ER, packaged in the Golgi apparatus, and transported to the plasma membrane via Golgi-derived secretory vesicles. Mobilization of reserves is also accompanied by dynamic membrane events. Our micrographs show that the surface monolayer of the lipid bodies fuses with the outer leaflet of the bilayer of protein-body membranes during the mobilization of lipid reserves. Following the breakdown of the protein reserves, the protein bodies assume a variety of configurations.Abbreviations ER endoplasmic reticulum - GA₃ gibberellic acid - P protoplasmic - E exoplasmic     

Membrane traffic from the endoplasmic reticulum to the Golgi apparatus is disturbed by an inhibitor of the Ca2+-ATPase in the ER

Summary An electron microscopic study of cress (Lepidium sativum L.) roots treated with cyclopiazonic acid (CPA), an inhibitor of the Ca²⁺-ATPase in the endoplasmic reticulum (ER) has been carried out. Drastic changes in the endomembrane system of the secretory root cap cells were observed. After treatment with CPA dense spherical or elliptoidal aggregates of ER (diameter 2–4 m) were formed in addition to the randomly distributed ER cisternae characteristic for control cells. The formation of ER aggregates indicates that in spite of an inhibition of the Ca²⁺ -ATPase in the ER by CPA, membrane synthesis in the ER continued. The ER aggregates are interpreted as a reservoir of ER membrane material newly synthesized during the 2 h CPA-treatment. Hypertrophied Golgi cisternae and secretory vesicles, which are characteristic for secretory cells under control conditions, were completely absent. Additionally the shape of the Golgi stacks was flat and the diameter of the cisternae was shortened by about one third. These phenomena are indicative of an inactive state of the Golgi apparatus. The cellular organization of both other cell types of the root cap, meristematic cells and statocytes, was not visibly affected by CPA, both having a relatively low secretory activity. The formation of ER aggregates as well as the reduction of Golgi compartments are indications for the existence of a unidirectional transport of membrane material from the ER to the Golgi. It is suggested that the membrane traffic from the ER to the Golgi apparatus is regulated by the cytosolic and/or luminal calcium concentration in secretory cells of the root cap.Abbreviations CPA cyclopiazonic acid - ER endoplasmic reticulum     

Analysis of COPII Vesicles Indicates a Role for the Emp47–Ssp120 Complex in Transport of Cell Surface Glycoproteins

Coat protein complex II (COPII) vesicle formation at the endoplasmic reticulum (ER) transports nascent secretory proteins forward to the Golgi complex. To further define the machinery that packages secretory cargo and targets vesicles to Golgi membranes, we performed a comprehensive proteomic analysis of purified COPII vesicles. In addition to previously known proteins, we identified new vesicle proteins including Coy1, Sly41 and Ssp120, which were efficiently packaged into COPII vesicles for trafficking between the ER and Golgi compartments. Further characterization of the putative calcium‐binding Ssp120 protein revealed a tight association with Emp47 and in emp47Δ cells Ssp120 was mislocalized and secreted. Genetic analyses demonstrated that EMP47 and SSP120 display identical synthetic positive interactions with IRE1 and synthetic negative interactions with genes involved in cell wall assembly. Our findings support a model in which the Emp47–Ssp120 complex functions in transport of plasma membrane glycoproteins through the early secretory pathway.      

Golgi Bypass: Skirting Around the Heart of Classical Secretion

Classical secretion consists of the delivery of transmembrane and soluble proteins to the plasma membrane and the extracellular medium, respectively, and is mediated by the organelles of the secretory pathway, the Endoplasmic Reticulum (ER), the ER exit sites, and the Golgi, as described by the Nobel Prize winner George Palade ( Palade 1975). At the center of this transport route, the Golgi stack has a major role in modifying, processing, sorting, and dispatching newly synthesized proteins to their final destinations. More recently, however, it has become clear that an increasing number of transmembrane proteins reach the plasma membrane unconventionally, either by exiting the ER in non-COPII vesicles or by bypassing the Golgi. Here, we discuss the evidence for Golgi bypass and the possible physiological benefits of it. Intriguingly, at least during Drosophila development, Golgi bypass seems to be mediated by a Golgi protein, dGRASP, which is found ectopically localized to the plasma membrane.The secretion of signal peptide-containing and transmembrane proteins through the cellular organelles that form the secretory pathway has been very well characterized over the years (Rothman 1994; Lee et al. 2004). During their translation, signal peptide-containing proteins are specifically recognized in the cytoplasm by the signal recognition particle and localize to the ER by virtue of the SRP binding its receptor (Nagai et al. 2003; Osborne et al. 2005). Other transmembrane proteins are embedded in the ER membrane by a posttranslational mechanism called C-tail anchoring by the GET complex (Schuldiner et al. 2008). Following transfer into or across the ER membrane, nascent proteins undergo folding, oligomerization, and addition of oligosaccharide chains followed by exit via specialized landmarks, known as ER exit sites (ERES) in mammalian cells and transitional ER (tER) sites in yeast and Drosophila. Both sites are characterized by the presence of cargo-containing coat protein complex II (COPII)-coated vesicles (Bonifacino and Glick 2004; Lee et al. 2004). Thereafter, most proteins are transported through the Golgi (in a manner that is still very much debated) before reaching their final destination, such as the plasma membrane for many transmembrane proteins and the extracellular medium for secreted proteins (Mellman and Warren 2000) (Fig. 1, red arrows).Open in a separate windowFigure 1.Classical trafficking, from the ER to the Golgi to the plasma membrane, is represented by the red arrows. A cargo protein can exit from an ERES in close proximity to the cis-Golgi (route 1a) or a peripheral ERES (route 1b), but irrespective of its ER exit, this protein follows a distinct pathway through the Golgi to the plasma membrane. This pathway is dependent on known SNARE proteins, NSF and SNAPs. As proteins pass from the ER and through the Golgi, their ER-derived high mannose oligosaccharides are modified by addition of complex sugars rendering these proteins EndoH-resistant. BFA treatment or loss of function of intra-Golgi SNAREs would lead to the retention of these proteins in the ER or Golgi and their diminished presence at the plasma membrane.Potential routes for Golgi bypass are represented by blue arrows. Like classical cargo proteins, Golgi bypass cargoes may exit from an ERES near the cis-Golgi (routes 2a,c) or a peripheral ERES (route 2b). However, the immediate fate of these proteins deviates from the classical pathway. A protein following route 2a (from an ERES near the cis-Golgi) or 2b (from a peripheral ERES) would traffic on ER-derived transport intermediates directly to the plasma membrane, routes perhaps taken by CD45 or αPS1. This route would require a specific set of SNAREs, yet to be identified. As these proteins do not pass through the Golgi stack, their high mannose N-glycans remain sensitive to EndoH. These pathways are also revealed by blocking passage through the Golgi either by the application of BFA, or by the loss of function of intra-Golgi SNAREs, (e.g., Syntaxin 5), and observing their continued transport to the plasma membrane. Proteins that follow route 2c would bypass the Golgi stack via an endosomal intermediate, which would facilitate their delivery to the plasma membrane via conventional endosomal fusion machinery. In the case of CFTR, its exit from the ER may occur from either ERES location to the TGN or endosomes. If it is directly delivered to endosomes, it is likely recycled back to the TGN in which the observed oligosaccharide modifications take place before reaching the plasma membrane.More recently, however, several examples of protein trafficking that deviate from this dogma have been discovered. First, an increasing number of cytoplasmic proteins (such as IL-1β, FGF2, MIF, and AcbA/Acb1) that do not harbor a signal peptide are found in the extracellular medium, and these display a wide range of critical activities. This “cytoplasmic protein unconventional secretion” has been extensively discussed elsewhere (Nickel and Seedorf 2008; Nickel and Rabouille 2009) and will not be covered in this volume, except for a brief note toward the end. Second, a small subset of proteins does not exit the ER by virtue of classical COPII-coated vesicles. Third, a few transmembrane proteins have been shown to reach the plasma membrane, bypassing the Golgi, which is the focus of this article.Why some proteins follow an unconventional route of secretion is intriguing but on the whole largely unknown. Through evolution, the cell has segregated processes within membrane compartments to maintain and optimize cellular functions. Why would mechanisms evolve to traffic a subset of proteins via unconventional routes? In this article, we discuss examples of Golgi bypass as well as outline why and how some proteins escape the conventional secretory pathway.     

Changes in the levels of seven proteins involved in polypeptide folding and transport during endosperm development of two barley genotypes differing in storage protein localisation

The Russian barley cultivar Nevsky lacks ₃ hordein and accumulates most of its hordein in the lumen of the endoplasmic reticulum and only a minor portion in the vacuole. In wild type barley and all other temperate cereals, storage proteins are deposited in the vacuole. F1 crosses revealed that the Nevsky phenotype is recessive; but the extent of hordein accumulation in the endoplasmic reticulum in F2 endosperm lacking ₃ hordein was very much less than in the Nevsky parent. In order to study the Nevsky endosperm phenotype we have measured the levels of seven proteins and two mRNAs involved in protein folding in the ER lumen or ER to Golgi transport during endosperm development. The protein levels were unaltered in Nevsky as compared to the wild-type variety Bomi. When the levels of these seven proteins were correlated with the rate of hordein accumulation, four of these (HSP70, PDI, Sar1p and Sec18p) were consistently up-regulated with hordein synthesis. Accumulation of hordein in the endoplasmic reticulum appears to be determined by the absence of ₃ hordein, or the product of a gene closely linked to it, plus one or more other recessive genes.     

Mutant SOD1 inhibits ER‐Golgi transport in amyotrophic lateral sclerosis

Cu/Zn‐superoxide dismutase is misfolded in familial and sporadic amyotrophic lateral sclerosis, but it is not clear how this triggers endoplasmic reticulum (ER) stress or other pathogenic processes. Here, we demonstrate that mutant SOD1 (mSOD1) is predominantly found in the cytoplasm in neuronal cells. Furthermore, we show that mSOD1 inhibits secretory protein transport from the ER to Golgi apparatus. ER‐Golgi transport is linked to ER stress, Golgi fragmentation and axonal transport and we also show that inhibition of ER‐Golgi trafficking preceded ER stress, Golgi fragmentation, protein aggregation and apoptosis in cells expressing mSOD1. Restoration of ER‐Golgi transport by over‐expression of coatomer coat protein II subunit Sar1 protected against inclusion formation and apoptosis, thus linking dysfunction in ER‐Golgi transport to cellular pathology. These findings thus link several cellular events in amyotrophic lateral sclerosis into a single mechanism occurring early in mSOD1 expressing cells.

     

Kinetically Distinct Sorting Pathways through the Golgi Exhibit Different Requirements for Arf1

To investigate the role of cytoplasmic sequences in directing transmembrane protein trafficking through the Golgi, we analyzed the sorting of VSV tsO45 G fusions with either the native G cytoplasmic domain (G) or an alternative cytoplasmic tail derived from the chicken AE1‐4 anion exchanger (G^AE). At restrictive temperature G^AE and G accumulated in the ER, and upon shifting the cells to permissive temperature both proteins folded and underwent transport through the Golgi. However, G^AE and G did not form hetero‐oligomers upon the shift to permissive temperature and they progressed through the Golgi with distinct kinetics. In addition, the transport of G through the proximal Golgi was Arf1 and COPI‐dependent, while G^AE progression through the proximal Golgi was Arf1 and COPI‐independent. Although Arf1 did not regulate the sorting of G^AE in the cis‐Golgi, Arf1 did regulate the exit of G^AE from the TGN. The trafficking of G^AE through the Golgi was similar to that of the native AE1‐4 anion exchanger, in that the progression of both proteins through the proximal Golgi was Arf1‐independent, while both required Arf1 to exit the TGN. We propose that the differential recognition of cytosolic signals in membrane‐spanning proteins by the Arf1‐dependent sorting machinery may influence the rate at which cargo progresses through the Golgi.      

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     

The Highly Conserved COPII Coat Complex Sorts Cargo from the Endoplasmic Reticulum and Targets It to the Golgi

Protein egress from the endoplasmic reticulum (ER) is driven by a conserved cytoplasmic coat complex called the COPII coat. The COPII coat complex contains an inner shell (Sec23/Sec24) that sorts cargo into ER-derived vesicles and an outer cage (Sec13/Sec31) that leads to coat polymerization. Once released from the ER, vesicles must tether to and fuse with the target membrane to deliver their protein and lipid contents. This delivery step also depends on the COPII coat, with coat proteins binding directly to tethering and regulatory factors. Recent findings have yielded new insight into how COPII-mediated vesicle traffic is regulated. Here we discuss the molecular basis of COPII-mediated ER–Golgi traffic, focusing on the surprising complexity of how ER-derived vesicles form, package diverse cargoes, and correctly target these cargoes to their destination.The port of entry into the secretory pathway is the endoplasmic reticulum (ER). Approximately one-third of the eukaryotic proteome traffics from this multifunctional organelle (Huh et al. 2003). This diverse set of cargo is translocated into the ER, folded, and modified before it travels to the Golgi, where further modifications occur. From the Golgi, cargo is sorted to other subcellular compartments to perform a variety of cellular functions. The highly conserved machinery required for these transport events was initially identified through genetic screens in the yeast Saccharomyces cerevisiae, and insights into the function of this machinery were provided through the use of in vitro transport assays. Advances in microscopy, in particular, the use of GFP fusion proteins and live cell imaging, have also played a critical role in understanding the dynamics of membrane traffic. In this article, we describe the mechanistic advances that have helped us to understand how diverse cargo correctly traffics from the ER to the Golgi complex in lower and higher eukaryotes. Even though these mechanisms are largely conserved, they are more complex at the molecular and organizational levels in metazoans.     

Protein Translocation across the Rough Endoplasmic Reticulum

The rough endoplasmic reticulum is a major site of protein biosynthesis in all eukaryotic cells, serving as the entry point for the secretory pathway and as the initial integration site for the majority of cellular integral membrane proteins. The core components of the protein translocation machinery have been identified, and high-resolution structures of the targeting components and the transport channel have been obtained. Research in this area is now focused on obtaining a better understanding of the molecular mechanism of protein translocation and membrane protein integration.Protein translocation across the rough endoplasmic reticulum (RER) is an ancient and evolutionarily conserved process that is analogous to protein export across the cytoplasmic membranes of eubacterial and archaebacterial cells both with respect to the mechanism and core components. The RER membrane of eukaryotic cells is contiguous with the nuclear envelope and is morphologically composed of interconnected cisternae and tubules. Electron microscope images of mammalian cells and tissues revealed that the cisternal regions of the cytoplasmic surface of the endoplasmic reticulum are densely studded by membrane-bound ribosomes (Palade 1955a,b), giving rise to the term “rough ER.” The RER-bound ribosomes in en face images are often arranged in spirals or hairpins (Palade 1955a; Christensen and Bourne 1999), indicative of polyribosomes that are actively engaged in protein translation.Consistent with this high density of membrane-bound ribosomes, the RER is a major site of protein biosynthesis in eukaryotic cells. The nuclear envelope, the Golgi, lysosome, peroxisome, plasma membrane, and endosomes are biosynthetically derived from the rough ER. The three major groups of proteins that are synthesized by RER-bound ribosomes include secretory proteins, integral membrane proteins destined for ER-derived membranes, and the lumenal-resident proteins of the ER, Golgi, nuclear envelope, and lysosome. For those membranes that are not physically linked to the ER (e.g., the lysosome), integral membrane and lumenal proteins are delivered to their destination by vesicular transport pathways. Bioinformatics analysis of fully sequenced eukaryotic genomes indicates that roughly 30% of open reading frames encode integral membrane proteins (Wallin and von Heijne 1998); hence, a major role of the RER is the biosynthesis of membrane proteins. An important class of membrane proteins that are integrated into the RER has single carboxy-terminal TM spans and are known as tail-anchored (TA) membrane proteins. The posttranslational integration pathway for TA proteins has been a subject of several recent reviews (Borgese and Fasana 2011; Shao and Hegde 2011), thus we will not address the TA pathway in this article.     

Vacuole biogenesis and protein transport to the plant vacuole: A comparison with the yeast vacuole and the mammalian lysosome

Summary The vacuole is often termed the lytic compartment of the plant cell. The yeast cell also possesses a vacuole containing acid hydrolases. In animal cells these enzymes are localized in the lysosome. Recent research suggests that there is good reason to regard these organelles as homologous in terms of protein transport. Although sorting motifs for the recognition of vacuolar proteins within the endomembrane system differ between the three organelles, there is an underlying similarity in targeting determinants in the cytoplasmic tails of Golgi-based receptors. In all three cases these determinants appear to interact with adaptins of clathrin-coated vesicles which ferry their cargo first of all to an endosomal compartment. The situation in sorting and targeting of plant vacuolar proteins is complicated by the fact that storage and lytic vacuoles may exist together in the same cell. The origin of these two types of vacuole is also a matter of some uncertanity.Abbrevations AP assembly protein - ALP alkaline phosphatase - ARF adenosine diphosphate ribosylation factor - BiP immunoglobulin binding protein - CCV clathrin coated vesicle - CPY carboxypeptidase-Y - DPAP dipeptidyl aminopeptidase - ER endoplasmic reticulum - GApp Golgi apparatus - LAMPs lysosomal associated membrane protein(s) - LAP lysosomal acid phosphatase - LIMPs lysosomal integral membrane protein(s) - MPRs mannosyl 6-phosphate receptors - MVB multivesicular bodies - NSF N-ethylmaleimide sensitive fusion (protein) - PAT phosphinotricine acetyltransferase - PB protein body - PHA phytohemagglutinin - PM plasma membrane - PSV protein storage vacuole - SNAPs soluble NSF attachment protein(s) - SNAREs SNAP receptor(s) - TGN trans Golgi network - TIP tonoplast integral protein - VPS vacuolar protein sorting - ZIO zinc iodide/osmium     

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.     

Glucosylceramide Biosynthesis is Involved in Golgi Morphology and Protein Secretion in Plant Cells

Lipids have an established role as structural components of membranes or as signalling molecules, but their role as molecular actors in protein secretion is less clear. The complex sphingolipid glucosylceramide (GlcCer) is enriched in the plasma membrane and lipid microdomains of plant cells, but compared to animal and yeast cells, little is known about the role of GlcCer in plant physiology. We have investigated the influence of GlcCer biosynthesis by glucosylceramide synthase (GCS) on the efficiency of protein transport through the plant secretory pathway and on the maintenance of normal Golgi structure. We determined that GlcCer is synthesized at the beginning of the plant secretory pathway [mainly endoplasmic reticulum (ER)] and that d ,l ‐threo‐1‐phenyl‐2‐decanoyl amino‐3‐morpholino‐propanol (PDMP) is a potent inhibitor of plant GCS activity in vitro and in vivo. By an in vivo confocal microscopy approach in tobacco leaves infiltrated with PDMP, we showed that the decrease in GlcCer biosynthesis disturbed the transport of soluble and membrane secretory proteins to the cell surface, as these proteins were partly retained intracellularly in the ER and/or Golgi. Electron microscopic observations of Arabidopsis thaliana root cells after high‐pressure freezing and freeze substitution evidenced strong morphological changes in the Golgi bodies, pointing to a link between decreased protein secretion and perturbations of Golgi structure following inhibition of GlcCer biosynthesis in plant cells.     

Degradation of transport-competent destabilized phaseolin with a signal for retention in the endoplasmic reticulum occurs in the vacuole

To understand how plant cells exert quality control over the proteins that pass through the secretory system we examined the transport and accumulation of the bean (Phaseolus vulgaris L.) vacuolar storage protein phaseolin, structurally modified to contain a helix-breaking epitope and carboxyterminal HDEL, an endoplasmic reticulum (ER)-retention signal. The constructs were expressed in tobacco (Nicotiana tabacum L.) with a seedspecific promoter. The results show that phaseolin-HDEL accumulates in the protein-storage vacuoles, indicating that HEDL does not contain sufficient information for retention in the ER. However, the ER of seeds expressing the phaseolin-HDEL construct contain relatively more phaseolin-HDEL compared to phaseolin in the ER of seeds expressing the phaseolin construct. This result indicates that the flow out of the ER is retarded but not arrested by the presence of HDEL. Introduction into phaseolin of the epitope himet (Hoffman et al., 1988, Plant Mol. Biol. 11, 717–729) greatly reduces the accumulation of HiMet phaseolin compared to normal phaseolin. However, the increased abundance within the ER is similar for both phaseolin-HDEL and HiMet phaseolin-HDEL. Using immunocytochemistry with specific antibodies, HiMet phaseolin was found in the ER, the Golgi stack, and in transport vesicles indicating that it was transport competent. It was also present at an early stage of seed development in the protein-storage vacuoles, but was not found there at later stages of seed development. Together these results support the conclusion that the HiMet epitope did not alter the structure of the protein sufficiently to make it transport incompetent. However, the protein was sufficiently destabilized to be degraded by vacuolar proteases.Abbreviations ER endoplasmic reticulum - BiP binding protein - IgG immunoglobulin G - M_r relative molecular mass The mention of vendor or product does not imply that they are endorsed or recommended by the US Department of Agriculture over vendors of similar products not mentionedThis work was supported by a grant from the National Science Foundation (Cell Biology) to M.J. Chrispeels and a fellowship from the Ministry of Education and Science, Spain-Fullbright Program to J.J. Pueyo. We thank H. Pelham for a gift of the constructs containing c-myc-SEKDEL and cmyc-FEHDEL and for a gift of anti-HDEL monoclonal antibodies. The original HiMet phaseolin construct was made by L. Hoffman and the phaseolin-HDEL or KDEL and HiMet-HDEL or KDEL constructs were made by D. Hunt as part of his doctoral research.     

Immunocytochemical evidence for intragranular processing of pro-opiomelanocortin in the melanotropic cells of the rabbit

Summary The immunogold technique, employing antisera with clear-cut specificities, was used to localise different processing stages of pro-opiomelanocortin (POMC) in rabbit melanotropic cells. While the antiserum against ₃-MSH labelled all the secretory granules including intrasaccular condensations in the Golgi apparatus, antisera against -MSH only labelled extra-Golgi secretory vesicles (SV). All extra-Golgi SV were likewise labelled with the three antisera against -MSH used, despite their different specificities for the desacetylated, N-acetylated or C-amidated forms of the peptide. The antibody against -endorphin also labelled the extra-Golgi SV, while only some SV were labelled with the antibody against -endorphin. These results correlate with biochemical data in favour of mainly — if not exclusively — intragranular processing of POMC. Except for ₃-MSH, the cleavage of which could coincide with Golgi packaging of secretory material, other post-translational modifications of the precursor seem to occur when SV are discharged outside the Golgi area. The cleavage of -endorphin appears to be a later step in POMC processing, occurring in some mature SV.     

Molecular Dissection of Erv26p Identifies Separable Cargo Binding and Coat Protein Sorting Activities

Efficient export of secretory alkaline phosphatase (ALP) from the endoplasmic reticulum depends on the conserved transmembrane sorting adaptor Erv26p/Svp26p. In the present study we investigated the mechanism by which Erv26p couples pro-ALP to the coat protein complex II (COPII) export machinery. Site-specific mutations were introduced into Erv26p, and mutant proteins were assessed in cell-free assays that monitor interactions with pro-ALP cargo and packaging into COPII vesicles. Mutations in the second and third loop domains of Erv26p inhibited interaction with pro-ALP, whereas mutations in the C-terminal tail sequence influenced incorporation into COPII vesicles and subcellular distribution. Interestingly mutations in the second loop domain also influenced Erv26p homodimer associations. Finally we demonstrated that Ktr3p, a cis-Golgi-localized mannosyltransferase, also relies on Erv26p for efficient COPII-dependent export from the endoplasmic reticulum. These findings demonstrate that Erv26p acts as a protein sorting adaptor for a variety of Type II transmembrane cargo proteins and requires domain-specific interactions with both cargo and coat subunits to promote efficient secretory protein transport.Anterograde transport in the eukaryotic secretory pathway is initiated by the formation of COPII²-coated vesicles that emerge from transitional ER sites. The COPII coat, which consists of the small GTPase Sar1p, Sec23/24 complex, and Sec13/31 complex, selects vesicle cargo through recognition of export signals and forms ER-derived vesicles through assembly of an outer layer cage structure (1, 2). Cytoplasmically exposed ER export signals have been identified in secretory cargo including the C-terminal dihydrophic and diacidic motifs (3, 4). Structural studies indicate that the Sec24p subunit of the COPII coat contains distinct binding sites for some of the molecularly defined export signals (5, 6). Thus a cycle of cargo-coat interactions regulated by the Sar1p GTPase directs anterograde movement of secretory proteins into ER-derived transport vesicles (7).Although many secretory proteins contain known export signals that interact directly with COPII subunits, the diverse array of secretory cargo that depends on this export route requires additional machinery for efficient collection of all cargo into COPII vesicles (1). For instance certain soluble secretory proteins as well as transmembrane cargo require protein sorting adaptors for efficient ER export. These membrane-spanning adaptors, or sorting receptors, interact directly with secretory cargo and with coat subunits to efficiently couple cargo to the COPII budding machinery. For example, ERGIC-53 acts as a protein sorting adaptor for several glycoproteins and has a large N-terminal lumenal domain that interacts with secretory proteins including blood coagulation factors, cathepsins, and α₁-antitrypsin (8–10). The cytoplasmic C-terminal tail of ERGIC-53 contains a diphenylalanine export signal that is necessary for COPII export as well as a dilysine motif required for COPI-dependent retrieval to the ER (11). Additional ER vesicle proteins identified in yeast have been shown to interact with the COPII coat as well as specific secretory proteins (12). For example Erv29p acts as a protein sorting adaptor for the soluble secretory proteins glyco-pro-α-factor and carboxypeptidase Y (13). Erv29p also contains COPII and COPI sorting signals that shuttle the protein between ER and Golgi compartments. More recently Erv26p was identified as a cargo receptor that escorts the pro-form of secretory alkaline phosphatase (ALP) into COPII-coated vesicles (14).Although COPII sorting receptors have been identified, the molecular mechanisms by which these receptors link cargo to coat remain poorly understood. Moreover it is not clear how cargo binding is regulated to promote interaction in the ER and then trigger dissociation in the Golgi complex. We have shown previously that Erv26p binds to pro-ALP and is required for efficient export of this secretory protein from the ER (14). Therefore specific lumenal regions of Erv26p are proposed to interact with pro-ALP, whereas cytosolically exposed sorting signals are presumably recognized and bound by coat subunits. To gain insight on the molecular contacts required for Erv26p sorting function, we undertook a systematic mutational analysis of this multispanning membrane protein. After generating a series of Erv26p mutants, we observed that mutation of specific residues in the third loop domain affect pro-ALP interaction and that residues in the C-terminal cytosolic tail are required for COPII and COPI transport. Finally mutation of residues in the second loop domain influenced Erv26p homodimer formation and sorting activity.     

Golgi‐mediated Glycosylation Determines Residency of the T2 RNase Rny1p in Saccharomyces cerevisiae

The role of glycosylation in the function of the T2 family of RNases is not well understood. In this work, we examined how glycosylation affects the progression of the T2 RNase Rny1p through the secretory pathway in Saccharomyces cerevisiae. We found that Rny1p requires entering into the ER first to become active and uses the adaptor protein Erv29p for packaging into COPII vesicles and transport to the Golgi apparatus. While inside the ER, Rny1p undergoes initial N‐linked core glycosylation at four sites, N37, N70, N103 and N123. Rny1p transport to the Golgi results in the further attachment of high‐glycans. Whereas modifications with glycans are dispensable for the nucleolytic activity of Rny1p, Golgi‐mediated modifications are critical for its extracellular secretion. Failure of Golgi‐specific glycosylation appears to direct Rny1p to the vacuole as an alternative destination and/or site of terminal degradation. These data reveal a previously unknown function of Golgi glycosylation in a T2 RNase as a sorting and secretion signal .      

Gibberellic-acid-stimulated Ca2+ accumulation in endoplasmic reticulum of barley aleurone: Ca2+ transport and steady-state levels

The steady-state levels of Ca²⁺ within the endoplasmic reticulum (ER) and the transport of ⁴⁵Ca²⁺ into isolated ER of barley (Hordeum vulgare L. cv. Himalaya) aleurone layers were studied. The Ca²⁺-sensitive dye indo-1. Endoplasmic reticulum was isolated and purified from indo-1-loaded protoplasts, and the Ca²⁺ level in the ER was measured using the Ca²⁺-sensitive dye indo-1. Endoplasmic reticulum was isolated and purified from indo-1-loaded protoplasts, and the Ca²⁺ level in the lumen of the ER was determined by the fluorescence-ratio method to be at least 3 M. Transport of ⁴⁵Ca²⁺ into the ER was studied in microsomal fractions isolated from aleurone layers incubated in the presence and absence of gibberellic acid (GA₃) and Ca²⁺. Isopycinic sucrose density gradient centrifugation of microsomal fractions isolated from aleurone layers or protoplasts separates ER from tonoplast and plasma membranes but not from the Golgi apparatus. Transport of ⁴⁵Ca²⁺ occurs primarily in the microsomal fraction enriched in ER and Golgi. Using monensin and heat-shock treatments to discriminate between uptake into the ER and Golgi, we established that ⁴⁵Ca²⁺ transport was into the ER. The sensitivity of ⁴⁵Ca²⁺ transport to inhibitors and the K_m of ⁴⁵Ca²⁺ uptake for ATP and Ca²⁺ transport in the microsomal fraction of barley aleurone cells. The rate of ⁴⁵Ca²⁺ transport is stimulated several-fold by treatment with GA₃. This effect of GA₃ is mediated principally by an effect on the activity of the Ca²⁺ transporter rather than on the amount of ER.Abbreviations CCR cytochrome-c reductase - DCCD dicyclohexylcarbodiimide - EGTA ethylene glycol bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - ER endoplasmic reticulum - FCCP carbonylcyanide p-trifluoromethoxyphenyl hydrazone - GA₃ gibberellic acid - IDPase inosine diphosphatase - Mon monensin