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The Transport Protein Particle (TRAPP) complexes are highly-conserved multisubunit complexes that act as nucleotide exchange factors (GEFs) for Rab GTPases. They act in both protein secretion and autophagy, and have also been proposed to have a role in other processes such as cytokinesis and ciliogenesis. There are two TRAPP complexes in metazoans: TRAPPII, which activates Rab11; and TRAPPIII, which activates Rab1. Both complexes share a core of small subunits that form the active site for the exchange of GDP for GTP. In addition, each TRAPP complex has distinct large subunits that determine the specificity of each complex towards its substrate Rab and are essential for activity in vivo. Crystal structures have revealed the organisation of the TRAPP core and the mechanism of Rab1 activation, whilst recent cryo-EM structures have unveiled the arrangement of the specific subunits around the core to form each complex. Combining these findings with functional experiments has allowed the proposal of mechanisms for how the specificity of each complex towards their cognate Rab is determined, and for the arrangement of these large complexes on the membrane.

In our 20

SummaryThe human genome encodes ∼20,000 proteins, many still uncharacterised. Scientific and social factors have resulted in a focus on well-studied proteins, leading to a concern that poorly understood genes are unjustifiably neglected. To address this, we have developed an “Unknome database” that ranks proteins based on how little is known about them. We applied RNAi in Drosophila to 260 unknown genes that are conserved between flies and humans. About a quarter are required for viability, and functional screening of the rest revealed hits for fertility, development, locomotion, protein quality control and resilience to stress. CRISPR/Cas9 gene disruption validated a component of Notch signalling and two genes contributing to male fertility. Our work demonstrates the importance of poorly understood genes, provides a resource for future research acceleration, and highlights a need for our awareness of ignorance to be protected from erosion by automated database annotation.

The Spike (S) protein of SARS-CoV-2 binds ACE2 to direct fusion with host cells. S comprises a large external domain, a transmembrane domain, and a short cytoplasmic tail. Understanding the intracellular trafficking of S is relevant to SARS-CoV-2 infection, and to vaccines expressing full-length S from mRNA or adenovirus vectors. Here we report a proteomic screen for cellular factors that interact with the cytoplasmic tail of S. We confirm interactions with the COPI and COPII vesicle coats, ERM family actin regulators, and the WIPI3 autophagy component. The COPII binding site promotes exit from the endoplasmic reticulum, and although binding to COPI should retain S in the early Golgi where viral budding occurs, there is a suboptimal histidine residue in the recognition motif. As a result, S leaks to the surface where it accumulates and can direct the formation of multinucleate syncytia. Thus, the trafficking signals in the tail of S indicate that syncytia play a role in the SARS-CoV-2 lifecycle.

The Golgi apparatus is an important site for the modification of most secreted and membrane proteins. Glycan processing is the major class of modification and is mediated by a large number of Golgi-resident glycosyltransferases and glycosidases. These Golgi enzymes are largely type II transmembrane domain (TMD) proteins consisting of a short N-terminal cytosolic tail, a relatively short TMD and a lumenal 'stem/stalk' region which acts as a spacer between the catalytic domain and the lipid bilayer. The cytosolic tail, TMD, and stem together make what is termed the CTS domain which is responsible for the specific localisation of these enzymes within sub-Golgi compartments via multiple mechanisms. In addition, the catalytic domains of some Golgi enzymes are secreted as a consequence of proteolytic cleavage within their TMDs or stem regions. Finally, there is evidence to suggest that when the retention of Golgi enzymes is perturbed they are targeted for lysosomal degradation.

The Golgi is the central sorting station in the secretory pathway and thus the destination of transport vesicles arriving from the endoplasmic reticulum and endosomes and from within the Golgi itself. Cell viability, therefore, requires that the Golgi accurately receives multiple classes of vesicle. One set of proteins proposed to direct vesicle arrival at the Golgi are the golgins, long coiled-coil proteins localized to specific parts of the Golgi stack. In mammalian cells, three of the golgins, TMF, golgin-84, and GMAP-210, can capture intra-Golgi transport vesicles when placed in an ectopic location. However, the individual golgins are not required for cell viability, and mouse knockout mutants only have defects in specific tissues. To further illuminate this system, we examine the Drosophila orthologs of these three intra-Golgi golgins. We show that ectopic forms can capture intra-Golgi transport vesicles, but strikingly, the cargo present in the vesicles captured by each golgin varies between tissues. Loss-of-function mutants show that the golgins are individually dispensable, although the loss of TMF recapitulates the male fertility defects observed in mice. However, the deletion of multiple golgins results in defects in glycosylation and loss of viability. Examining the vesicles captured by a particular golgin when another golgin is missing reveals that the vesicle content in one tissue changes to resemble that of a different tissue. This reveals a plasticity in Golgi organization between tissues, providing an explanation for why the Golgi is sufficiently robust to tolerate the loss of many of the individual components of its membrane traffic machinery.

Glycosylation is a diverse and abundant modification of proteins, lipids and RNA. The fidelity of glycosylation is, in part, assured by the correct compartmentalisation of Golgi-resident glycosylation enzymes within the Golgi stack. The COPI adaptor GOLPH3 has been shown to interact with the cytoplasmic tails of a subset of Golgi enzymes and direct their retention in the Golgi. However, other mechanisms of retention, and other roles for GOLPH3, have been proposed, and a comprehensive characterisation of the clientele of GOLPH3 and its paralogue GOLPH3L has been lacking. The role of GOLPH3 is of particular interest as it is frequently amplified in several solid tumour types. Here, we combine two orthogonal proteomic analyses to identify a diverse range of GOLPH3+3L clients and find that they act in a wide spectrum of glycosylation pathways, or have other roles in the Golgi. Using binding studies, bioinformatics and an in vivo Golgi retention assay, we show that GOLPH3+3L interact with the cytoplasmic tails of their clients through membrane-proximal positively-charged residues. Furthermore, deletion of GOLPH3+3L causes diverse defects in glycosylation. Thus, GOLPH3+3L are major COPI adaptors that impinge on most, if not all, of the glycosylation pathways of the Golgi.

The TRAPP complexes are nucleotide exchange factors that play essential roles in membrane traffic and autophagy. TRAPPII activates Rab11, and TRAPPIII activates Rab1, with the two complexes sharing a core of small subunits that affect nucleotide exchange but being distinguished by specific large subunits that are essential for activity in vivo. Crystal structures of core subunits have revealed the mechanism of Rab activation, but how the core and the large subunits assemble to form the complexes is unknown. We report a cryo-EM structure of the entire Drosophila TRAPPIII complex. The TRAPPIII-specific subunits TRAPPC8 and TRAPPC11 hold the catalytic core like a pair of tongs, with TRAPPC12 and TRAPPC13 positioned at the joint between them. TRAPPC2 and TRAPPC2L link the core to the two large arms, with the interfaces containing residues affected by disease-causing mutations. The TRAPPC8 arm is positioned such that it would contact Rab1 that is bound to the core, indicating how the arm could determine the specificity of the complex. A lower resolution structure of TRAPPII shows a similar architecture and suggests that the TRAPP complexes evolved from a single ur-TRAPP.

SummaryThe TRAPP complexes are highly conserved nucleotide exchange factors, with TRAPPIII activating Rab1 and TRAPPII acting primarily on Rab11. The two complexes share a core of small subunits that affect nucleotide exchange, but are distinguished by additional large subunits that are essential for activity in vivo, and are mutated in a range of human disorders. Crystal structures of the core subunits have revealed the mechanism of Rab activation, but how and why the large subunits associate with the core remains unclear. We report here a cryo-EM structure of the entire TRAPPIII complex fromDrosophila. The TRAPPIII-specific subunits TRAPPC8 and TRAPPC11 hold the catalytic core like a pair of tongs, with TRAPPC12 and TRAPPC13 positioned at the joint between them. TRAPPC2 and TRAPPC2L link the core to the two large arms, with the interfaces containing residues affected by disease-causing mutations. The TRAPPC8 arm is positioned such that it would contact bound Rab1, indicating how the arms could alter the Rab specificity of the core. A lower resolution structure of TRAPPII shows a similar architecture, and suggests that the TRAPP complexes evolved from a single ur-TRAPP.

The Spike (S) protein of SARS-CoV-2 binds ACE2 to direct fusion with host cells. S comprises a large external domain, a transmembrane domain (TMD) and a short cytoplasmic tail. Understanding the intracellular trafficking of S is relevant to SARS-CoV-2 infection, and to vaccines expressing full-length S from mRNA or adenovirus vectors. We have applied proteomics to identify cellular factors that interact with the cytoplasmic tail of S. We confirmed interactions with the COPI and COPII vesicle coats, ERM family actin regulators, and the WIPI3 autophagy component. The COPII binding-site promotes exit from the endoplasmic reticulum (ER), and although COPI-binding should retain S in the early Golgi where viral budding occurs, there is a suboptimal histidine residue in the recognition motif. As a result, S leaks to the surface where it accumulates and can direct the formation of multinucleate syncytia. Thus, the trafficking signals in the tail of S indicate that syncytia play a role in the SARS-CoV-2 lifecycle.

The internal organisation of the cell depends on tethers at destination organelles to selectively capture incoming transport vesicles to facilitate SNARE-mediated fusion. The golgin long coiled-coil proteins function as tethers that contributes to this specificity at the Golgi (1). Golgin-97, golgin-245 and GCC88 golgins of the trans-Golgi capture vesicles derived from endosomes, which serve to recycle the critical Golgi machinery required to deliver lysosomal hydrolases and to maintain exocytosis. Retrograde trafficking from endosomes to the trans-Golgi network (TGN) is a complex process that involves the sorting of transmembrane cargo proteins into distinct transport vesicles by adaptors from multiple pathways. The content of these distinct vesicles, which golgin they target and the factors that mediate this targeting are not well understood. The major challenges that have limited advances in these areas is the transient nature of vesicle tethering, and the redundancies in their mechanisms that confound experimental dissection. To gain better insight into these problems, we performed organelle proteomics using the Localisation of Organelle Proteins by Isotope Tagging after Differential ultraCentrifugation (LOPIT-DC) method on a system in which an ectopic golgin causes vesicles to accumulate in a tethered state (2). By incorporating Bayesian statistical modelling into our analysis (3), we determined that over 45 transmembrane proteins and 51 peripheral membrane proteins of the endosomal network are on vesicles captured by golgin-97, including known cargo and components of the clathrin/AP-1, retromer-dependent and -independent transport pathways. We also determined a distinct class of vesicles shared by golgin-97, golgin-245 and GCC88 that is enriched in TMEM87A, a multi-pass transmembrane protein of unknown function that has previously been implicated in endosome-to-Golgi retrograde transport (4). Finally, we categorically demonstrate that the vesicles that these golgins capture are retrograde transport vesicles based on the lack of enrichment of lysosomal hydrolases in our LOPIT-DC data, and from correlative light electron tomography images of spherical vesicles captured by golgin-97. Together, our study demonstrates the power of combining LOPIT-DC with Bayesian statistical analysis in interrogating the dynamic spatial movement of proteins in transport vesicles.

Summary Pathogenic bacteria enter the cytosol of host cells through uptake into bacteria-containing vacuoles (BCVs) and subsequent rupture of the vacuolar membrane [1]. Bacterial invaders are sensed either directly, through cytosolic pattern-recognition receptors specific for bacterial ligands, or indirectly, through danger receptors that bind host molecules displayed in an abnormal context, for example, glycans on damaged BCVs [2, 3, 4]. In contrast to damage caused by Listeria monocytogenes, a Gram-positive bacterium, BCV rupture by Gram-negative pathogens such as Shigella flexneri or Salmonella Typhimurium remains incompletely understood [5, 6]. The latter may cause membrane damage directly, when inserting their Type Three Secretion needles into host membranes, or indirectly through translocated bacterial effector proteins [7, 8, 9]. Here, we report that sphingomyelin, an abundant lipid of the luminal leaflet of BCV membranes, and normally absent from the cytosol, becomes exposed to the cytosol as an early predictive marker of BCV rupture by Gram-negative bacteria. To monitor subcellular sphingomyelin distribution, we generated a live sphingomyelin reporter from Lysenin, a sphingomyelin-specific toxin from the earthworm Eisenia fetida [10, 11]. Using super resolution live imaging and correlative light and electron microscopy (CLEM), we discovered that BCV rupture proceeds through two distinct successive stages: first, sphingomyelin is gradually translocated into the cytosolic leaflet of the BCV, invariably followed by cytosolic exposure of glycans, which recruit galectin-8, indicating bacterial entry into the cytosol. Exposure of sphingomyelin on BCVs may therefore act as an early danger signal alerting the cell to imminent bacterial invasion., Graphical Abstract, Highlights • Lysenin serves as a reporter of sphingomyelin exposure in the mammalian cytosol • Chemical-, toxin-, or pathogen-induced membrane damage exposes sphingomyelin • Sphingomyelin exposure precedes catastrophic breakage of bacteria-containing vacuoles • Cytosolic sphingomyelin is indicative of membrane stress and imminent pathogen entry, Ellison et al. show that the pore-forming toxin Lysenin can be used as a reporter for detecting sphingomyelin in the cytosolic leaflet of cellular membranes. The breakout of Gram-negative bacteria from vacuoles into the host cytosol follows a precisely choreographed process in which sphingomyelin exposure precedes catastrophic membrane damage.

The GTPases of the Ras superfamily regulate cell growth, membrane traffic and the cytoskeleton, and a wide range of diseases are caused by mutations in particular members. They function as switchable landmarks with the active GTP-bound form recruiting to the membrane a specific set of effector proteins. The GTPases are precisely controlled by regulators that promote acquisition of GTP (GEFs) or its hydrolysis to GDP (GAPs). We report here MitoID, a method for identifying effectors and regulators by performing in vivo proximity biotinylation with mitochondrially-localized forms of the GTPases. Applying this to 11 human Rab GTPases identified many known effectors and GAPs, as well as putative novel effectors, with examples of the latter validated for Rab2, Rab5 and Rab9. MitoID can also efficiently identify effectors and GAPs of Rho and Ras family GTPases such as Cdc42, RhoA, Rheb, and N-Ras, and can identify GEFs by use of GDP-bound forms.

The Golgi apparatus lies at the centre of the secretory pathway. It consists of a series of flattened compartments typically organised into a stack that, in mammals, is connected to additional stacks to form a Golgi ribbon. The Golgi is responsible for the maturation and modification of proteins and lipids, and receives and exports vesicles to and from multiple destinations within the cell. This complex trafficking network requires that only the correct vesicles fuse with the correct destination membrane. Recently, a group of coiled-coil proteins called golgins were shown to not only capture incoming vesicles but to also provide specificity to the tethering step. This raises many interesting questions about how they interact with other components of membrane traffic, some of which may also contribute to specificity.

The use of Drosophila melanogaster as a model organism has been pivotal to understanding the developmental processes of metazoans. However, the use of flies for studying subcellular organization is hampered by a paucity of reliable reagents to label specific organelles. Here, we describe the generation of mouse monoclonal antibodies against a set of markers of the secretory and endocytic pathways, along with goat polyclonal antibodies against two Golgi proteins. We show that the monoclonal antibodies are highly specific and sufficiently sensitive to detect endogenous proteins in crude extracts by immunoblotting with little background staining. By immunofluorescence the major compartments of the membrane traffic system (including the endoplasmic reticulum, the Golgi, and early and late endosomes) are labeled by at least one antibody. Moreover, the antibodies can be used to label organelles in fly tissues including salivary glands and wing imaginal discs. We anticipate that these antibodies will provide a useful tool kit to facilitate the investigation of how the endomembrane system functions and varies in the diverse tissue types of metazoans., Summary: We report the generation and characterization of set of monoclonal and polyclonal antibodies for labeling the major compartments of the secretory and endocytic pathways in Drosophila melanogaster.

All cells contain numerous membrane-bound organelles that carry out specific functions. These compartments do not, however, act in isolation. Some are in direct contact via membrane contact sites, while others exchange material via specific vesicles or tubular carriers laden with cargo. The term tethering in the context of this review is used to describe the primary recognition and docking of transport carriers with acceptor organelles that occurs before SNARE engagement and membrane fusion. However, it is important to note that other tethering events occur, for example, between organelles in direct contact, which do not lead to fusion.

A third of yeast genes encode for proteins that function in the endomembrane system. However, the precise localization for many of these proteins is still uncertain. Here, we visualized a collection of ~500 N-terminally, green fluorescent protein (GFP), tagged proteins of the yeast Saccharomyces cerevisiae. By co-localizing them with seven known markers of endomembrane compartments we determined the localization for over 200 of them. Using this approach, we create a systematic database of the various secretory compartments and identify several new residents. Focusing in, we now suggest that Lam5 resides in contact sites between the Endoplasmic Reticulum and the late Golgi. Additionally, analysis of interactions between the COPI coat and co-localizing proteins from our screen identifies a subset of proteins that are COPI-cargo. In summary, our approach defines the protein roster within each compartment enabling characterization of the physical and functional organization of the endomembrane system and its components.

Muschalik and Munro introduce golgins and their roles as vesicle tethers and scaffolds at the Golgi.

Summary The GTPase Arf1 is the major regulator of vesicle traffic at both the cis- and trans-Golgi. Arf1 is activated at the cis-Golgi by the guanine nucleotide exchange factor (GEF) GBF1 and at the trans-Golgi by the related GEF BIG1 or its paralog, BIG2. The trans-Golgi-specific targeting of BIG1 and BIG2 depends on the Arf-like GTPase Arl1. We find that Arl1 binds to the dimerization and cyclophilin binding (DCB) domain in BIG1 and report a crystal structure of human Arl1 bound to this domain. Residues in the DCB domain that bind Arl1 are required for BIG1 to locate to the Golgi in vivo. DCB domain-binding residues in Arl1 have a distinct conformation from those in known Arl1-effector complexes, and this plasticity allows Arl1 to interact with different effectors of unrelated structure. The findings provide structural insight into how Arf1 GEFs, and hence active Arf1, achieve their correct subcellular distribution., Graphical Abstract, Highlights • Arl1 binds the N-terminal DCB domain of the Arf1-GEF BIG1 • Structure of the human DCBBIG1/Arl1 complex is solved at 2.3 Å resolution • The Arl1 binding surface on the DCB domain is unrelated to known Arl1 effectors • Structure-guided mutation shows BIG1 needs Ar11 binding for Golgi targeting, The GTPase Arf1 is essential for Golgi function and is activated on the trans-Golgi by the BIG1 exchange factor. A second GTPase, Arl1, is required for BIG1 recruitment. Galindo et al. find that Arl1 binds to the DCB domain of BIG1 and determine the structure of human Arl1 bound to this domain.

In yeast, the TRAPP complexes activate Rab1 with TRAPPII also activating Rab11, but less is known about the two TRAPPs in metazoans. Riedel et al. show that in Drosophila melanogaster, TRAPPIII is an essential Rab1 activator, and TRAPPII activates Rab1 and Rab11 and becomes essential when an unrelated Rab11 activator is deleted., Originally identified in yeast, transport protein particle (TRAPP) complexes are Rab GTPase exchange factors that share a core set of subunits. TRAPPs were initially found to act on Ypt1, the yeast orthologue of Rab1, but recent studies have found that yeast TRAPPII can also activate the Rab11 orthologues Ypt31/32. Mammals have two TRAPP complexes, but their role is less clear, and they contain subunits that are not found in the yeast complexes but are essential for cell growth. To investigate TRAPP function in metazoans, we show that Drosophila melanogaster have two TRAPP complexes similar to those in mammals and that both activate Rab1, whereas one, TRAPPII, also activates Rab11. TRAPPII is not essential but becomes so in the absence of the gene parcas that encodes the Drosophila orthologue of the SH3BP5 family of Rab11 guanine nucleotide exchange factors (GEFs). Thus, in metazoans, Rab1 activation requires TRAPP subunits not found in yeast, and Rab11 activation is shared by TRAPPII and an unrelated GEF that is metazoan specific.

The specificity of membrane traffic involves tethers at destination organelles that selectively capture incoming transport vesicles to allow SNAREs on opposing membranes to then assemble and drive fusion. Tethers include both protein complexes and long coiled-coil proteins, although how they contribute to specificity remains unclear. The golgin coiled-coil proteins at the Golgi apparatus capture vesicles from different origins, but the vesicle-specific molecular cues that they recognize are unknown. Vesicle tethering is typically a transient process and therefore is challenging to interrogate in vivo. Thus, we have used a system in which an ectopic golgin causes vesicles to accumulate in a tethered state. By applying proximity biotinylation to the golgin-captured vesicles, we identify TBC1D23, an apparently catalytically inactive member of a family of Rab GTPase-activating proteins (GAPs), as a vesicle-golgin adaptor that is required for endosome-to-Golgi trafficking. The Rab GAP domain of TBC1D23 binds to a conserved motif at the tip of golgin-245 and golgin-97 at the trans-Golgi, while the C terminus binds to the WASH complex on endosome-derived vesicles. Thus, TBC1D23 is a specificity determinant that links the vesicle to the target membrane during endosome-to-Golgi trafficking.

Summary The Rab GTPases recruit peripheral membrane proteins to intracellular organelles. These Rab effectors typically mediate the motility of organelles and vesicles and contribute to the specificity of membrane traffic. However, for many Rabs, few, if any, effectors have been identified; hence, their role remains unclear. To identify Rab effectors, we used a comprehensive set of Drosophila Rabs for affinity chromatography followed by mass spectrometry to identify the proteins bound to each Rab. For many Rabs, this revealed specific interactions with Drosophila orthologs of known effectors. In addition, we found numerous Rab-specific interactions with known components of membrane traffic as well as with diverse proteins not previously linked to organelles or having no known function. We confirm over 25 interactions for Rab2, Rab4, Rab5, Rab6, Rab7, Rab9, Rab18, Rab19, Rab30, and Rab39. These include tethering complexes, coiled-coiled proteins, motor linkers, Rab regulators, and several proteins linked to human disease., Graphical Abstract, Highlights • Proteomic screen identifies effectors of Drosophila Rabs with a human ortholog • Specific hits include orthologs of numerous known effectors of mammalian Rabs • Validated effectors include traffic proteins and those of unknown function • Orthologs of disease genes CLEC16A, LRRK2, and SPG20 are validated as effectors, Rab GTPases organize cellular compartments by recruiting specific effectors to organelle membranes. This paper describes affinity chromatography using all Drosophila Rabs with a mammalian ortholog. The Rab interactors found include known effectors, tethering complexes, coiled-coil proteins, motor proteins, proteins of unknown function, and several proteins linked to human disease.

BACKGROUND: The internal organization of cells depends on mechanisms to ensure that transport carriers, such as vesicles, fuse only with the correct destination organelle. Several types of proteins have been proposed to confer specificity to this process, and we have recently shown that a set of coiled-coil proteins on the Golgi, called golgins, are able to capture specific classes of carriers when relocated to an ectopic location. RESULTS: Mapping of six different golgins reveals that, in each case, a short 20-50 residue region is necessary and sufficient to capture specific carriers. In all six of GMAP-210, golgin-84, TMF, golgin-97, golgin-245, and GCC88, this region is located at the extreme N-terminus of the protein. The vesicle-capturing regions of GMAP-210, golgin-84, and TMF capture intra-Golgi vesicles and share some sequence features, suggesting that they act in a related, if distinct, manner. In the case of GMAP-210, this shared feature is in addition to a previously characterized "amphipathic lipid-packing sensor" motif that can capture highly curved membranes, with the two motifs being apparently involved in capturing distinct types of vesicles. Of the three GRIP domain golgins that capture endosome-to-Golgi carriers, golgin-97 and golgin-245 share a closely related capture motif, whereas that in GCC88 is distinct, suggesting that it works by a different mechanism and raising the possibility that the three golgins capture different classes of endosome-derived carriers that share many cargos but have distinct features for recognition at the Golgi. CONCLUSIONS: For six different golgins, the capture of carriers is mediated by a short region at the N-terminus of the protein. There appear to be at least four different types of motif, consistent with specific golgins capturing specific classes of carrier and implying the existence of distinct receptors present on each of these different carrier classes.

The Golgi apparatus is the central organelle in the secretory pathway and plays key roles in glycosylation, protein sorting, and secretion in plants. Enzymes involved in the biosynthesis of complex polysaccharides, glycoproteins, and glycolipids are located in this organelle, but the majority of them remain uncharacterized. Here, we studied the Arabidopsis (Arabidopsis thaliana) membrane proteome with a focus on the Golgi apparatus using localization of organelle proteins by isotope tagging. By applying multivariate data analysis to a combined data set of two new and two previously published localization of organelle proteins by isotope tagging experiments, we identified the subcellular localization of 1,110 proteins with high confidence. These include 197 Golgi apparatus proteins, 79 of which have not been localized previously by a high-confidence method, as well as the localization of 304 endoplasmic reticulum and 208 plasma membrane proteins. Comparison of the hydrophobic domains of the localized proteins showed that the single-span transmembrane domains have unique properties in each organelle. Many of the novel Golgi-localized proteins belong to uncharacterized protein families. Structure-based homology analysis identified 12 putative Golgi glycosyltransferase (GT) families that have no functionally characterized members and, therefore, are not yet assigned to a Carbohydrate-Active Enzymes database GT family. The substantial numbers of these putative GTs lead us to estimate that the true number of plant Golgi GTs might be one-third above those currently annotated. Other newly identified proteins are likely to be involved in the transport and interconversion of nucleotide sugar substrates as well as polysaccharide and protein modification.

Specificity in Arf1 GEF recruitment to the trans-Golgi, and thus in localized Arf1 activation, is provided by an Arf-like G protein., The small G protein Arf1 regulates Golgi traffic and is activated by two related types of guanine nucleotide exchange factor (GEF). GBF1 acts at the cis-Golgi, whereas BIG1 and its close paralog BIG2 act at the trans-Golgi. Peripheral membrane proteins such as these GEFs are often recruited to membranes by small G proteins, but the basis for specific recruitment of Arf GEFs, and hence Arfs, to Golgi membranes is not understood. In this paper, we report a liposome-based affinity purification method to identify effectors for small G proteins of the Arf family. We validate this with the Drosophila melanogaster Arf1 orthologue (Arf79F) and the related class II Arf (Arf102F), which showed a similar pattern of effector binding. Applying the method to the Arf-like G protein Arl1, we found that it binds directly to Sec71, the Drosophila ortholog of BIG1 and BIG2, via an N-terminal region. We show that in mammalian cells, Arl1 is necessary for Golgi recruitment of BIG1 and BIG2 but not GBF1. Thus, Arl1 acts to direct a trans-Golgi–specific Arf1 GEF, and hence active Arf1, to the trans side of the Golgi.

The integral membrane protein Atg9 is delivered to the autophagosome in yeast and mammalian cells. We find that Atg9 does not originate from mitochondria and cannot reach the autophagosome directly from the ER. Instead, pairwise combinations of mutations in Golgi-endosomal traffic components cause defects in Atg9 delivery during starvation., While many of the proteins required for autophagy have been identified, the source of the membrane of the autophagosome is still unresolved with the endoplasmic reticulum (ER), endosomes, and mitochondria all having been evoked. The integral membrane protein Atg9 is delivered to the autophagosome during starvation and in the related cytoplasm-to-vacuole (Cvt) pathway that occurs constitutively in yeast. We have examined the requirements for delivery of Atg9-containing membrane to the yeast autophagosome. Atg9 does not appear to originate from mitochondria, and Atg9 cannot reach the forming autophagosome directly from the ER or early Golgi. Components of traffic between Golgi and endosomes are known to be required for the Cvt pathway but do not appear required for autophagy in starved cells. However, we find that pairwise combinations of mutations in Golgi-endosomal traffic components apparently only required for the Cvt pathway can cause profound defects in Atg9 delivery and autophagy in starved cells. Thus it appears that membrane that contains Atg9 is delivered to the autophagosome from the Golgi-endosomal system rather than from the ER or mitochondria. This is underestimated by examination of single mutants, providing a possible explanation for discrepancies between yeast and mammalian studies on Atg9 localization and autophagosome formation.

Summary The various membranes of eukaryotic cells differ in composition, but it is at present unclear if this results in differences in physical properties. The sequences of transmembrane domains (TMDs) of integral membrane proteins should reflect the physical properties of the bilayers in which they reside. We used large datasets from both fungi and vertebrates to perform a comprehensive comparison of the TMDs of proteins from different organelles. We find that TMDs are not generic but have organelle-specific properties with a dichotomy in TMD length between the early and late parts of the secretory pathway. In addition, TMDs from post-ER organelles show striking asymmetries in amino acid compositions across the bilayer that is linked to residue size and varies between organelles. The pervasive presence of organelle-specific features among the TMDs of a particular organelle has implications for TMD prediction, regulation of protein activity by location, and sorting of proteins and lipids in the secretory pathway., Graphical Abstract Highlights ► Transmembrane domains (TMDs) vary in length and residue composition between organelles ► TMD lengths differ pre- versus post-Golgi but not between apical and basolateral surfaces ► The differences between TMDs are large enough to have value in predicting location ► Pervasive differences mean TMDs could collectively contribute to membrane properties

Genetic screens in the yeast Saccharomyces cerevisiae have identified many proteins involved in the secretory pathway, most of which have orthologues in higher eukaryotes. To investigate whether there are additional proteins that are required for secretion in metazoans but are absent from yeast, we used genome-wide RNA interference (RNAi) to look for genes required for secretion of recombinant luciferase from Drosophila S2 cells. This identified two novel components of the secretory pathway that are conserved from humans to plants. Gryzun is distantly related to, but distinct from, the Trs130 subunit of the TRAPP complex but is absent from S. cerevisiae. RNAi of human Gryzun (C4orf41) blocks Golgi exit. Kish is a small membrane protein with a previously uncharacterised orthologue in yeast. The screen also identified Drosophila orthologues of almost 60% of the yeast genes essential for secretion. Given this coverage, the small number of novel components suggests that contrary to previous indications the number of essential core components of the secretory pathway is not much greater in metazoans than in yeasts.

Vesicles and other carriers destined for the Golgi apparatus must be guided to the correct cisternae. Golgins, long coiled-coil proteins that localize to particular Golgi subdomains via their C termini, are candidate regulators of vesicle sorting. In this study, we report that the GRIP domain golgins, whose C termini bind the Arf-like 1 G protein on the trans-Golgi, can also bind four members of the Rab family of G proteins. The Rab2-, Rab6-, Rab19-, and Rab30-binding sites are within the coiled-coil regions that are not required for Golgi targeting. Binding sites for two of these Rabs are also present on two coiled-coil proteins of the cis-Golgi, the Drosophila melanogaster orthologues of GM130 and GMAP-210. We suggest an integrated model for a tentacular Golgi in which coiled-coil proteins surround the Golgi to capture and retain Rab-containing membranes, excluding other structures such as ribosomes. Binding sites for diverse Rabs could ensure that incoming carriers are captured on first contact and moved to their correct destination within the stack.

Small G proteins play a central role in the organization of the secretory and endocytic pathways. The majority of such small G proteins are members of the Rab family, which are anchored to the bilayer by C-terminal prenyl groups. However, the recruitment of some effectors, including vesicle coat proteins, is mediated by a second class of small G proteins that is unique in having an N-terminal amphipathic helix that becomes available for membrane insertion upon GTP binding. Sar1, Arf1, and Arf6 are the best-characterized members of this ADP-ribosylation factor (Arf) family. In addition, all eukaryotes contain additional distantly related G proteins, often called Arf like, or Arls. The complete Arf family in humans has 29 members. The roles of these related G proteins are poorly understood, but recent work has shown that some are involved in membrane traffic or organizing the cytoskeleton. Here we review what is known about all the members of the Arf family, along with the known regulatory molecules that convert them between GDP- and GTP-bound states.

The small G proteins of the Arf family play critical roles in membrane trafficking and cytoskeleton organization. However, the function of some members of the family remains poorly understood including Arl5 which is widely conserved in eukaryotes. Humans have two closely related Arl5 paralogues (Arl5a and Arl5b), and both Arl5a and Arl5b localize to the trans-Golgi with Arl5b being involved in retrograde traffic from endosomes to the Golgi apparatus. To investigate the function of Arl5, we have used Drosophila melanogaster as a model system. We find that the single Arl5 orthologue in Drosophila also localizes to the trans-Golgi, but flies lacking the Arl5 gene are viable and fertile. By using both liposome and column based affinity chromatography methods we find that Arl5 interacts with the Golgi-associated retrograde protein (GARP) complex that acts in the tethering of vesicles moving from endosomes to the trans-Golgi network (TGN). In Drosophila tissues the GARP complex is partially displaced from the Golgi when Arl5 is absent, and the late endosomal compartment is enlarged. In addition, in HeLa cells GARP also becomes cytosolic upon depletion of Arl5b. These phenotypes are consistent with a role in endosome-to-Golgi traffic, but are less severe than loss of GARP itself. Thus it appears that Arl5 is one of the factors that directs the recruitment of the GARP complex to the trans-Golgi, and this function is conserved in both flies and humans.

The GTPase Arl3p is required to recruit a second GTPase, Arl1p, to the Golgi in Saccharomyces cerevisiae. Arl1p binds to the GRIP domain, which is present in a number of long coiled-coil proteins or 'golgins'. Here we show that Arl3p is not myristoylated like most members of the Arf family, but is instead amino-terminally acetylated by the NatC complex. Targeting of Arl3p also requires a Golgi membrane protein Sys1p. The human homologues of Arl3p (Arf-related protein 1 (ARFRP1)) and Sys1p (hSys1) can be isolated in a complex after chemical cross-linking. This suggests that the targeting of ARFRP1/Arl3p to the Golgi is mediated by a direct interaction between its acetylated N terminus and Sys1p/hSys1.

Golgins are large coiled-coil proteins that play a role in Golgi structure and vesicle traffic. The Arf-like GTPase Arl1 regulates the translocation of GRIP domain-containing golgins to Golgi membranes. We report here the 1.7 A resolution structure of human Arl1-GTP in a complex with the GRIP domain of golgin-245. The structure reveals that the GRIP domain consists of an S-shaped arrangement of three helices. The domain forms a homodimer that binds two Arl1-GTPs using two helices from each monomer. The structure is consistent with golgin-245 forming parallel coiled-coils and suggests how Arl1-GTP/GRIP complexes interact with Golgi membranes via the N termini of Arl1-GTP and the C-terminal tails of the GRIP domains. In cells, bivalent association with Arl1-GTP would increase residence time of the golgins on Golgi membranes. Despite no conservation of sequence, topology, or even helical direction, several other effectors form similar interactions with small GTPases via a pair of alpha helices, suggesting a common structural basis for effector recognition.

The yeast Saccharomyces cerevisiae is widely regarded as being only capable of producing N-linked glycans with high-mannose structures. To investigate the glycan structures made in different mutant strains, we made use of a reporter protein consisting of a version of hen egg lysozyme that contains a single site for N-linked glycosylation. Mass spectrometry analysis of the attached glycans revealed that a large proportion contained an unexpected extra mass corresponding to a single N-acetylhexosamine residue. In addition, the glycosylated lysozyme was recognized by an N-acetylglucosamine specific lectin. The genome of S. cerevisiae contains an uncharacterized open reading frame, YOR320c, that is related to a known N-acetylglucosaminyltransferase. Deletion of this ORF resulted in the disappearance of the extra mass on the N-linked glycans and loss of lectin binding. We show that the protein encoded by YOR320c (which we term Gnt1p) is localized to the Golgi apparatus and has GlcNAc-transferase activity in vitro. The physiological role of Gnt1p is unclear because mutants lacking the protein show no obvious growth or cell wall defects. Nonetheless, these results indicate that heterologous glycoproteins expressed in yeast can receive N-glycans with structures other than high mannose. In addition, they indicate that the lumen of the yeast Golgi contains UDP-GlcNAc, which may facilitate reconstitution of higher eukaryotic N-glycan processing.

In the version of Supplementary Table 1 originally published with this Article, in the sheet relating to Fig. 3c, all values in the ‘golgin-97-mito’ column were 1.3 times larger than the actual values, which was due to author error when generating the Supplementary Table. These errors did not affect the graph in Fig. 3c, which was plotted with the correct values. Supplementary Table 1 has now been replaced so that it contains the correct values.