ERS-24, a Mammalian v-SNARE Implicated in Vesicle Traffic between the ER and the Golgi

We report the identification and characterization of ERS-24 (Endoplasmic Reticulum SNARE of 24 kD), a new mammalian v-SNARE implicated in vesicular transport between the ER and the Golgi. ERS24 is incorporated into 20S docking and fusion particles and disassembles from this complex in an ATP-dependent manner. ERS-24 has significant sequence homology to Sec22p, a v-SNARE in Saccharomyces cerevisiae required for transport between the ER and the Golgi. ERS-24 is localized to the ER and to the Golgi, and it is enriched in transport vesicles associated with these organelles.Newly formed transport vesicles have to be selectively targeted to their correct destinations, implying the existence of a set of compartment-specific proteins acting as unique receptor–ligand pairs. Such proteins have now been identified (Söllner et al., 1993a ; Rothman, 1994): one partner efficiently packaged into vesicles, termed a v-SNARE,¹ and the other mainly localized to the target compartment, a t-SNARE. Cognate pairs of v- and t-SNAREs, capable of binding each other specifically, have been identified for the ER–Golgi transport step (Lian and Ferro-Novick, 1993; Søgaard et al., 1994), the Golgi–plasma membrane transport step (Aalto et al., 1993; Protopopov et al., 1993; Brennwald et al., 1994) in Saccharomyces cerevisiae, and regulated exocytosis in neuronal synapses (Söllner et al., 1993a ; for reviews see Scheller, 1995; Südhof, 1995). Additional components, like p115, rab proteins, and sec1 proteins, appear to regulate vesicle docking by controlling the assembly of SNARE complexes (Søgaard et al., 1994; Lian et al., 1994; Sapperstein et al., 1996; Hata et al., 1993; Pevsner et al., 1994).In contrast with vesicle docking, which requires compartment-specific components, the fusion of the two lipid bilayers uses a more general machinery derived, at least in part, from the cytosol (Rothman, 1994), which includes an ATPase, the N-ethylmaleimide–sensitive fusion protein (NSF) (Block et al., 1988; Malhotra et al., 1988), and soluble NSF attachment proteins (SNAPs) (Clary et al., 1990; Clary and Rothman, 1990; Whiteheart et al., 1993). Only the assembled v–t-SNARE complex provides high affinity sites for the consecutive binding of three SNAPs (Söllner et al., 1993b ; Hayashi et al., 1995) and NSF. When NSF is inactivated in vivo, v–t-SNARE complexes accumulate, confirming that NSF is needed for fusion after stable docking (Søgaard et al., 1994).The complex of SNAREs, SNAPs, and NSF can be isolated from detergent extracts of cellular membranes in the presence of ATPγS, or in the presence of ATP but in the absence of Mg²⁺, and sediments at ∼20 Svedberg (20S particle) (Wilson et al., 1992). In the presence of MgATP, the ATPase of NSF disassembles the v–t-SNARE complex and also releases SNAPs. It seems likely that this step somehow initiates fusion.To better understand vesicle flow patterns within cells, it is clearly of interest to identify new SNARE proteins. Presently, the most complete inventory is in yeast, but immunolocalization is difficult in yeast compared with animal cells, and many steps in protein transport have been reconstituted in animal extracts (Rothman, 1992) that have not yet been developed in yeast. Therefore, it is important to create an inventory of SNARE proteins in animal cells. The most unambiguous and direct method for isolating new SNAREs is to exploit their ability to assemble together with SNAPs and NSF into 20S particles and to disassemble into subunits when NSF hydrolyzes ATP. Similar approaches have already been successfully used to isolate new SNAREs implicated in ER to Golgi (Søgaard et al., 1994) and intra-Golgi transport (Nagahama et al., 1996), in addition to the original discovery of SNAREs in the context of neurotransmission (Söllner et al., 1993a ).Using this method, we now report the isolation and detailed characterization of ERS-24 (Endoplasmic Reticulum SNARE of 24 kD), a new mammalian v-SNARE that is localized to the ER and Golgi. ERS-24 is found in transport vesicles associated with the transitional areas of the ER and with the rims of Golgi cisternae, suggesting a role for ERS-24 in vesicular transport between these two compartments.     

The Structural Architecture of an Infectious Mammalian Prion Using Electron Cryomicroscopy

The structure of the infectious prion protein (PrP^Sc), which is responsible for Creutzfeldt-Jakob disease in humans and bovine spongiform encephalopathy, has escaped all attempts at elucidation due to its insolubility and propensity to aggregate. PrP^Sc replicates by converting the non-infectious, cellular prion protein (PrP^C) into the misfolded, infectious conformer through an unknown mechanism. PrP^Sc and its N-terminally truncated variant, PrP 27–30, aggregate into amorphous aggregates, 2D crystals, and amyloid fibrils. The structure of these infectious conformers is essential to understanding prion replication and the development of structure-based therapeutic interventions. Here we used the repetitive organization inherent to GPI-anchorless PrP 27–30 amyloid fibrils to analyze their structure via electron cryomicroscopy. Fourier-transform analyses of averaged fibril segments indicate a repeating unit of 19.1 Å. 3D reconstructions of these fibrils revealed two distinct protofilaments, and, together with a molecular volume of 18,990 ų, predicted the height of each PrP 27–30 molecule as ~17.7 Å. Together, the data indicate a four-rung β-solenoid structure as a key feature for the architecture of infectious mammalian prions. Furthermore, they allow to formulate a molecular mechanism for the replication of prions. Knowledge of the prion structure will provide important insights into the self-propagation mechanisms of protein misfolding.     

Yif1B Is Involved in the Anterograde Traffic Pathway and the Golgi Architecture

Yif1B is an intracellular membrane‐bound protein belonging to the Yip family, shown previously to control serotonin 5‐HT_1A receptor targeting to dendrites. Because some Yip proteins are involved in the intracellular traffic between the ER and the Golgi, here we investigated the precise localization of Yif1B in HeLa cells. We found that Yif1B is not resident into the Golgi, but rather belongs to the IC compartment. After analyzing the role of Yif1B in protein transport, we showed that the traffic of the VSVG protein marker was accelerated in Yif1B depleted HeLa cells, as well as in hippocampal neurons from Yif1B KO mice. Conversely, Yif1B depletion in HeLa cells did not change the retrograde traffic of ShTx. Interestingly, in long term depletion of Yif1B as in Yif1B KO mice, we observed a disorganized Golgi architecture in CA1 pyramidal hippocampal neurons, which was confirmed by electron microscopy. However, because short term depletion of Yif1B did not change Golgi structure, it is likely that the implication of Yif1B in anterograde traffic does not rely on its role in structural organization of the Golgi apparatus, but rather on its shuttling between the ER, the IC and the Golgi compartments.      

The Mammalian Calcium-binding Protein, Nucleobindin (CALNUC), Is a Golgi Resident Protein

We have identified CALNUC, an EF-hand, Ca²⁺-binding protein, as a Golgi resident protein. CALNUC corresponds to a previously identified EF-hand/calcium-binding protein known as nucleobindin. CALNUC interacts with Gα_i3 subunits in the yeast two-hybrid system and in GST-CALNUC pull-down assays. Analysis of deletion mutants demonstrated that the EF-hand and intervening acidic regions are the site of CALNUC's interaction with Gα_i3. CALNUC is found in both cytosolic and membrane fractions. The membrane pool is tightly associated with the luminal surface of Golgi membranes. CALNUC is widely expressed, as it is detected by immunofluorescence in the Golgi region of all tissues and cell lines examined. By immunoelectron microscopy, CALNUC is localized to cis-Golgi cisternae and the cis-Golgi network (CGN). CALNUC is the major Ca²⁺-binding protein detected by ⁴⁵Ca²⁺-binding assay on Golgi fractions. The properties of CALNUC and its high homology to calreticulin suggest that it may play a key role in calcium homeostasis in the CGN and cis-Golgi cisternae.     

Mammalian Oocytes Locally Remodel Follicular Architecture to Provide the Foundation for Germline-Soma Communication

1. Download high-res image (146KB)
2. Download full-size image

     

Architecture of the Golgi apparatus of a scale-forming alga: biogenesis and transport of scales

Mechanisms of transport of secretory products across the Golgi apparatus (GA) as well as of scale formation in prymnesiophytes have remained controversial. We have used a quantitative morphological approach to study formation and transport of scales across the GA in haploid cells of Pleurochrysis sp. The GA of these cells differs from the GA of higher plants in at least six morphological characteristics. Our results show that scales form in the trans-Golgi network (TGN) and transit the TGN in heretofore unrecognized prosecretory vesicles. Prosecretory vesicles differentiate into secretory vesicles prior to exocytosis of scales to the cell surface. Because prosecretory vesicles are only fragments of TGN cisternae, the classical model of cisternal progression is not a valid mechanism of transport in this alga. TGN transport vesicles are also involved in scale formation; however, the role of tubular connections between cisternae of a single stack-TGN unit is not clear. The relationship of two morphological types of cisternal dilations to a membrane-associated, bottlebrush-shaped macromolecule of novel morphology suggests a new hypothesis for the biogenesis of scales.     

Camillo Golgi and the discovery of the Golgi apparatus

 Camillo Golgi (1843–1926) was born at Corteno, near Brescia, in northern Italy. After graduating in Medicine at the ancient University of Pavia, the former seat of great scientists and naturalists, Golgi continued a long-standing Italian tradition by studying the histology of the nervous system. While working as a modest physician at Abbiategrasso, a small town near Pavia, he developed a silver–osmium technique, the ”reazione nera” (black reaction), for which he was awarded the Nobel Prize in 1906. In the late 1890’s, 25 years after the publication of his black reaction and while Professor of General Pathology in Pavia, Golgi noticed a fine internal network in only partially silver-osmium-blackened Purkinje cells. Following confirmation by his assistant Emilio Veratti, Golgi published the discovery, called the ”apparato reticolare interno”, in the Bollettino della Società medico-chirurgica di Pavia in 1898, which is now considered the birthday of the ”Golgi apparatus”. The discovery of the Golgi apparatus can be added to the long list of accidental discoveries. The man after whom it is named was not a cytologist engaged in studying the inner structure of the cell, but a pathologist searching to prove a neuroanatomical theory. Accepted: 24 October 1997     

Mitosis controls the Golgi and the Golgi controls mitosis

In mammals, the Golgi complex is structured in the form of a continuous membranous system composed of up to 100 stacks connected by tubular bridges, the 'Golgi ribbon'. During mitosis, the Golgi undergoes extensive fragmentation through a multistage process that allows its correct partitioning and inheritance by daughter cells. Strikingly, this Golgi fragmentation is required not only for inheritance but also for mitotic entrance itself, since its block results in the arrest of the cell cycle in G2. This is called the 'Golgi mitotic checkpoint'. Recent studies have identified the severing of the ribbon into its constituent stacks during early G2 as the precise stage of Golgi fragmentation that controls mitotic entry. This opens new ways to elucidate the mechanism of the Golgi checkpoint.     

The Golgi apparatus: defining the identity of Golgi membranes

The Golgi apparatus is a stack of compartments that serves as a central junction for membrane traffic, with carriers moving through the stack as well as arriving from, and departing toward, many other destinations in the cell. This requires that the different compartments in the Golgi recruit from the cytosol a distinct set of proteins to mediate accurate membrane traffic. This recruitment appears to reflect recognition of small GTPases of the Rab and Arf family, or of lipid species such as PtdIns(4)P and diacylglycerol, which provide a unique "identity" for each compartment. Recent work is starting to reveal the mechanisms by which these labile landmarks are generated in a spatially restricted manner on specific parts of the Golgi.     

The Golgi apparatus and cell polarity: Roles of the cytoskeleton,the Golgi matrix,and Golgi membranes

Membrane trafficking plays a crucial role in cell polarity by directing lipids and proteins to specific subcellular locations in the cell and sustaining a polarized state. The Golgi apparatus, the master organizer of membrane trafficking, can be subdivided into three layers that play different mechanical roles: a cytoskeletal layer, the so-called Golgi matrix, and the Golgi membranes. First, the outer regions of the Golgi apparatus interact with cytoskeletal elements, mainly actin and microtubules, which shape, position, and orient the organelle. Closer to the Golgi membranes, a matrix of long coiled–coiled proteins not only selectively captures transport intermediates but also participates in signaling events during polarization of membrane trafficking. Finally, the Golgi membranes themselves serve as active signaling platforms during cell polarity events. We review here the recent findings that link the Golgi apparatus to cell polarity, focusing on the roles of the cytoskeleton, the Golgi matrix, and the Golgi membranes.     

Characterization of clofibrate-induced retrograde Golgi membrane movement to the endoplasmic reticulum: clofibrate distinguishes the Golgi from the trans Golgi network

Clofibrate-induced retrograde Golgi membrane movement was blocked or retarded when NRK cells were treated with sodium azide/2-deoxyglucose, nocodazole, taxol, and destruxin B, indicating that it depends on energy, and the dynamic state of microtubules, and being acidic or vacuolar-type ATPase function. PDMP and phospholipase A2 inhibitors also blocked it. These characteristics are similar to those of brefeldin A (BFA) and nordihydroguaiaretic acid (NDGA), inducers of retrograde Golgi membrane movement. However, clofibrate was distinguished from BFA in that BFA action was insensitive to phospholipase A2 inhibitors and from NDGA in that NDGA stabilized microtubules against nocodazole and its action was almost insensitive to taxol. The trans Golgi network (TGN) was resistant to clofibrate, while BFA and NDGA dispersed it. To our knowledge, clofibrate is the first drug to show such different effects on the Golgi and TGN and, therefore, is expected to be a useful tool to distinguish their architecture and/or membrane dynamics.     

Characterization of Clofibrate-induced Retrograde Golgi Membrane Movement to the Endoplasmic Reticulum: Clofibrate Distinguishes the Golgi from the Trans Golgi Network

Clofibrate-induced retrograde Golgi membrane movement was blocked or retarded when NRK cells were treated with sodium azide/2-deoxyglucose, nocodazole, taxol, and destruxin B, indicating that it depends on energy, and the dynamic state of microtubules, and being acidic or vacuolar-type ATPase function. PDMP and phospholipase A₂ inhibitors also blocked it. These characteristics are similar to those of brefeldin A (BFA) and nordihydroguaiaretic acid (NDGA), inducers of retrograde Golgi membrane movement. However, clofibrate was distinguished from BFA in that BFA action was insensitive to phospholipase A₂ inhibitors and from NDGA in that NDGA stabilized microtubules against nocodazole and its action was almost insensitive to taxol. The trans Golgi network (TGN) was resistant to clofibrate, while BFA and NDGA dispersed it. To our knowledge, clofibrate is the first drug to show such different effects on the Golgi and TGN and, therefore, is expected to be a useful tool to distinguish their architecture and/or membrane dynamics.     

WHAMMing into the Golgi

A new paper from Campellone et al. in a recent issue of Cell identifies WHAMM, a multifunctional protein that stimulates Arp2/3-mediated actin polymerization, binds and organizes microtubules, and influences the structure and efficiency of the Golgi complex. WHAMM's membrane localization at the entry face of the Golgi complex is novel for an actin nucleation-promoting factor, and highlights the importance of the cytoskeleton in organizing the secretory pathway.     

Motoring around the Golgi

The Golgi apparatus is a dynamic organelle through which nascent secretory and transmembrane proteins are transported, post-translationally modified and finally packaged into carrier vesicles for transport along the cytoskeleton to a variety of destinations. In the past decade, studies have shown that a number of 'molecular motors' are involved in maintaining the proper structure and function of the Golgi apparatus. Here, we review just some of the many functions performed by these mechanochemical enzymes - dyneins, kinesins, myosins and dynamin - in relation to the Golgi apparatus.     

Conserved oligomeric Golgi complex specifically regulates the maintenance of Golgi glycosylation machinery

Cell surface lectin staining, examination of Golgi glycosyltransferases stability and localization, and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis were employed to investigate conserved oligomeric Golgi (COG)-dependent glycosylation defects in HeLa cells. Both Griffonia simplicifolia lectin-II and Galanthus nivalus lectins were specifically bound to the plasma membrane glycoconjugates of COG-depleted cells, indicating defects in activity of medial- and trans-Golgi-localized enzymes. In response to siRNA-induced depletion of COG complex subunits, several key components of Golgi glycosylation machinery, including MAN2A1, MGAT1, B4GALT1 and ST6GAL1, were severely mislocalized. MALDI-TOF analysis of total N-linked glycoconjugates indicated a decrease in the relative amount of sialylated glycans in both COG3 KD and COG4 KD cells. In agreement to a proposed role of the COG complex in retrograde membrane trafficking, all types of COG-depleted HeLa cells were deficient in the Brefeldin A- and Sar1 DN-induced redistribution of Golgi resident glycosyltransferases to the endoplasmic reticulum. The retrograde trafficking of medial- and trans-Golgi-localized glycosylation enzymes was affected to a larger extent, strongly indicating that the COG complex regulates the intra-Golgi protein movement. COG complex-deficient cells were not defective in Golgi re-assembly after the Brefeldin A washout, confirming specificity in the retrograde trafficking block. The lobe B COG subcomplex subunits COG6 and COG8 were localized on trafficking intermediates that carry Golgi glycosyltransferases, indicating that the COG complex is directly involved in trafficking and maintenance of Golgi glycosylation machinery.     

Positioning the Golgi apparatus

Ríos et al. (2004) report in this issue that the Golgi protein GMAP-210 is sufficient to confer pericentrosomal positioning and recruits gamma-tubulin and associated microtubule-nucleating ring complex proteins to Golgi membranes. The results raise the possibility that short microtubules emanate from the Golgi to mediate its organization and positioning.     

Spectrins and the Golgi

Several isoforms of spectrin membrane skeleton proteins have been localized to the Golgi complex. Golgi-specific membrane skeleton proteins associate with the Golgi as a detergent-resistant cytoskeletal structure that likely undergoes a dynamic assembly process that accommodates Golgi membrane dynamics. This review discusses the potential roles for this molecule in Golgi functions. In particular, it will focus on a recently identified distant cousin to conventional erythroid spectrin variously named Syne-1, Nesprin, myne, Enaptin, MSP-300, and Ank-1. Syne-1 has the novel ability to bind to both the Golgi and the nuclear envelope, a property that raises several intriguing and novel insights into Golgi structure and function. These include (1) the facilitation of interactions between Golgi and transitional ER sites on the nuclear envelope of muscle cells, and (2) an ability to impart localized specificity to the secretory pathway within large multinucleate syncytia such as skeletal muscle fibers.     

Exiting the Golgi complex

The composition and identity of cell organelles are dictated by the flux of lipids and proteins that they receive and lose through cytosolic exchange and membrane trafficking. The trans-Golgi network (TGN) is a major sorting centre for cell lipids and proteins at the crossroads of the endocytic and exocytic pathways; it has a complex dynamic structure composed of a network of tubular membranes that generate pleiomorphic carriers targeted to different destinations. Live-cell imaging combined with three-dimensional tomography has recently provided the temporal and topographical framework that allows the assembly of the numerous molecular machineries so far implicated in sorting and trafficking at the TGN.