Regulation of the nucleocytoplasmic trafficking of viral and cellular proteins by ubiquitin and small ubiquitin-related modifiers

Nucleocytoplasmic trafficking of many cellular proteins is regulated by nuclear import/export signals as well as post-translational modifications such as covalent conjugation of ubiquitin and small ubiquitin-related modifiers (SUMOs). Ubiquitination and SUMOylation are rapid and reversible ways to modulate the intracellular localisation and function of substrate proteins. These pathways have been co-opted by some viruses, which depend on the host cell machinery to transport their proteins in and out of the nucleus. In this review, we will summarise our current knowledge on the ubiquitin/SUMO-regulated nuclear/subnuclear trafficking of cellular proteins and describe examples of viral exploitation of these pathways.     

Disturbed vesicular trafficking of membrane proteins in prion disease

The pathogenic mechanism of prion diseases remains unknown. We recently reported that prion infection disturbs post-Golgi trafficking of certain types of membrane proteins to the cell surface, resulting in reduced surface expression of membrane proteins and abrogating the signal from the proteins. The surface expression of the membrane proteins was reduced in the brains of mice inoculated with prions, well before abnormal symptoms became evident. Prions or pathogenic prion proteins were mainly detected in endosomal compartments, being particularly abundant in recycling endosomes. Some newly synthesized membrane proteins are delivered to the surface from the Golgi apparatus through recycling endosomes, and some endocytosed membrane proteins are delivered back to the surface through recycling endosomes. These results suggest that prions might cause neuronal dysfunctions and cell loss by disturbing post-Golgi trafficking of membrane proteins via accumulation in recycling endosomes. Interestingly, it was recently shown that delivery of a calcium channel protein to the cell surface was impaired and its function was abrogated in a mouse model of hereditary prion disease. Taken together, these results suggest that impaired delivery of membrane proteins to the cell surface is a common pathogenic event in acquired and hereditary prion diseases.     

Glycosylation affects the protein stability and cell surface expression of Kv1.4 but Not Kv1.1 potassium channels. A pore region determinant dictates the effect of glycosylation on trafficking

Kv1.1 and Kv1.4 potassium channels are plasma membrane glycoproteins involved in action potential repolarization. We have shown previously that glycosylation affects the gating function of Kv1.1 and that a pore region determinant of Kv1.1 and Kv1.4 affects their cell surface trafficking negatively or positively, respectively. Here we investigated the role of N-glycosylation of Kv1.1 and Kv1.4 on their protein stability, cellular localization pattern, and trafficking to the cell surface. We found that preventing N-glycosylation of Kv1.4 decreased its protein stability, induced its high partial intracellular retention, and decreased its cell surface protein levels, whereas it had little or no effect on these parameters for Kv1.1. Exchanging a trafficking pore region determinant between Kv1.1 and Kv1.4 reversed these effects of glycosylation on these chimeric channels. Thus it appeared that the Kv1.4 pore region determinant and the sugar tree attached to the S1-S2 linker showed some type of dependence in promoting proper trafficking of the protein to the cell surface, and this dependence can be transferred to chimeric Kv1.1 proteins that contain the Kv1.4 pore. Understanding the different trafficking programs of Kv1 channels, and whether they are altered by glycosylation, will highlight the different posttranslational mechanisms available to cells to modify their cell surface ion channel levels and possibly their signaling characteristics.     

Dissecting the molecular function of reggie/flotillin proteins

Reggie-1/flotillin-2 and reggie-2/flotillin-1 are ubiquitously expressed, well-conserved proteins that are associated with membrane microdomains known as rafts. Studies from us and others have suggested a role in various cellular processes such as insulin signaling, T cell activation, membrane trafficking, phagocytosis, and epidermal growth factor receptor signaling. Recent findings also demonstrate that reggie-1 is associated with cell motility and transformation. However, the exact function of reggie proteins remains to be clarified. In this review, we will focus on some recent findings that have shed new light on the elusive molecular function of these highly interesting proteins. We will especially discuss the emerging role of reggie proteins in membrane receptor signaling and membrane trafficking, with emphasis on the regulation of the molecular function of reggies by post-translational modifications such as phosphorylation and lipid modifications.     

Actin in membrane trafficking

Actin cytoskeleton remodeling provides the forces required for a variety of cellular processes based on membrane dynamics, such as endocytosis, exocytosis, and vesicular trafficking at the Golgi. All these events are coordinated by networks of associated proteins, and some of them are functionally connected with cell migration. The site and the duration of actin polymerization, in connection with vesicle budding and fusion, are tightly controlled by both small GTPases and the large GTPase dynamin. Recent advances in the understanding of the mechanisms coupling actin dynamics with membrane trafficking at the cell surface have been brought by the combined studies of actin polymerizing factors and of the endocytic/exocytic machinery.     

Plasma membrane targeting of exocytic SNARE proteins

SNARE proteins play a central role in the process of intracellular membrane fusion. Indeed, the interaction of SNAREs present on two opposing membranes is generally believed to provide the driving force to initiate membrane fusion. Eukaryotic cells express a large number of SNARE isoforms, and the function of individual SNAREs is required for specific intracellular fusion events. Exocytosis, the fusion of secretory vesicles with the plasma membrane, employs the proteins syntaxin and SNAP-25 as plasma membrane SNAREs. As a result, exocytosis is dependent upon the targeting of these proteins to the plasma membrane; however, the mechanisms that underlie trafficking of exocytic syntaxin and SNAP-25 proteins to the cell surface are poorly understood. The intracellular trafficking itinerary of these proteins is particularly intriguing as syntaxins are tail-anchored (or Type IV) membrane proteins, whereas SNAP-25 is anchored to membranes via a central palmitoylated domain-there is no common consensus for the trafficking of such proteins within the cell. In this review, we discuss the plasma membrane targeting of these essential exocytic SNARE proteins.     

Chaperone-mediated specificity in Ras and Rap signaling

Abstract

Ras and Rap proteins are closely related small guanosine triphosphatase (GTPases) that share similar effector-binding domains but operate in a very different signaling networks; Ras has a dominant role in cell proliferation, while Rap mediates cell adhesion. Ras and Rap proteins are regulated by several shared processes such as post-translational modification, phosphorylation, activation by guanine exchange factors and inhibition by GTPase-activating proteins. Sub-cellular localization and trafficking of these proteins to and from the plasma membrane are additional important regulatory features that impact small GTPases function. Despite its importance, the trafficking mechanisms of Ras and Rap proteins are not completely understood. Chaperone proteins play a critical role in trafficking of GTPases and will be the focus of the discussion in this work. We will review several aspects of chaperone biology focusing on specificity toward particular members of the small GTPase family. Understanding this specificity should provide key insights into drug development targeting individual small GTPases.     

Understanding the distinct subcellular trafficking of CD36 and GLUT4 during the development of myocardial insulin resistance

CD36 and GLUT4 are the main cardiac trans-sarcolemmal transporters for long-chain fatty acids and glucose, respectively. Together they secure the majority of cardiac energy demands. Moreover, these transporters each represent key governing kinetic steps in cardiac fatty acid and glucose fluxes, thereby offering major sites of regulation. The underlying mechanism of this regulation involves a perpetual vesicle-mediated trafficking (recycling) of both transporters between intracellular stores (endosomes) and the cell surface. In the healthy heart, CD36 and GLUT4 translocation to the cell surface is under short-term control of the same physiological stimuli, most notably increased contraction and insulin secretion. However, under chronic lipid overload, a condition that accompanies a Western lifestyle, CD36 and GLUT4 recycling are affected distinctly, with CD36 being expelled to the sarcolemma while GLUT4 is imprisoned within the endosomes. Moreover, the increased CD36 translocation towards the cell surface is a key early step, setting the heart on a route towards insulin resistance and subsequent contractile dysfunction. Therefore, the proteins making up the trafficking machinery of CD36 need to be identified with special focus to the differences with the protein composition of the GLUT4 trafficking machinery. These proteins that are uniquely dedicated to either CD36 or GLUT4 traffic may offer targets to rectify aberrant substrate uptake seen in the lipid-overloaded heart. Specifically, CD36-dedicated trafficking regulators should be inhibited, whereas such GLUT4-dedicated proteins would need to be activated. Recent advances in the identification of CD36-dedicated trafficking proteins have disclosed the involvement of vacuolar-type H⁺-ATPase and of specific vesicle-associated membrane proteins (VAMPs). In this review, we summarize these recent findings and sketch a roadmap of CD36 and GLUT4 trafficking compatible with experimental findings.     

Large-scale identification of Legionella pneumophila Dot/Icm substrates that modulate host cell vesicle trafficking pathways

The bacterial pathogen Legionella pneumophila replicates in a specialized vacuole within host cells. Establishment of the replication vacuole depends on the Dot/Icm translocation system that delivers a large number of protein substrates into the host cell. The functions of most substrates are unknown. Here, we analysed a defined set of 127 confirmed or candidate Dot/Icm substrates for their effect on host cell processes using yeast as a model system. Expression of 79 candidates caused significant yeast growth defects, indicating that these proteins impact essential host cell pathways. Notably, a group of 21 candidates interfered with the trafficking of secretory proteins to the yeast vacuole. Three candidates that caused yeast secretory defects (SetA, Ceg19 and Ceg9) were investigated further. These proteins impinged upon vesicle trafficking at distinct stages and had signals that allowed translocation into host cells by the Dot/Icm system. Ectopically produced SetA, Ceg19 and Ceg9 localized to secretory organelles in mammalian cells, consistent with a role for these proteins in modulating host cell vesicle trafficking. Interestingly, the ability of SetA to cause yeast phenotypes was dependent upon a functional glycosyltransferase domain. We hypothesize that SetA may glycosylate a component of the host cell vesicle trafficking machinery during L. pneumophila infection.     

Endocytic traffic in polarized epithelial cells: role of the actin and microtubule cytoskeleton

The cytoskeleton is required for multiple cellular events including endocytosis and the transfer of cargo within the endocytic system. Polarized epithelial cells are capable of endocytosis at either of their distinct apical or basolateral plasma membrane domains. Actin plays a role in internalization at both cell surfaces. Microtubules and actin are required for efficient transcytosis and delivery of proteins to late endosomes and lysosomes. Microtubules are also important in apical recycling pathways and, in some polarized cell types, basolateral recycling requires actin. The microtubule motor proteins dynein and kinesin and the class I unconventional myosin motors play a role in many of these trafficking steps. This review examines the endocytic pathways of polarized epithelial cells and focuses on the emerging roles of the actin cytoskeleton in these processes.     

The EGF receptor: a nexus for trafficking and signaling

Ligand binding to the EGF receptor initiates both the activation of mitogenic signal transduction pathways plus trafficking events that relocalize the receptor on the cell surface and within intracellular compartments. The trafficking compartments include caveolae, clathrin-coated pits, and various endosome populations prior to receptor degradation in lysosomes. Evidence is presented that distinct signaling pathways are initiated from these different compartments. These include the Ras/MAP kinase cascade and the PLC-dependent hydrolysis of PI-4,5 P(2). Multiple tyrosine kinase substrates that facilitate EGF receptor trafficking between these various compartments, as well as the participation of phosphoinositides and Ras-like G proteins in the trafficking pathway are also described.     

Connecting vesicular transport with lipid synthesis: FAPP2

Next to the protein-based machineries composed of small G-proteins, coat complexes, SNAREs and tethering factors, the lipid-based machineries are emerging as important players in membrane trafficking. As a component of these machineries, lipid transfer proteins have recently attracted the attention of cell biologists for their involvement in trafficking along different segments of the secretory pathway. Among these, the four-phosphate adaptor protein 2 (FAPP2) was discovered as a protein that localizes dynamically with the trans-Golgi network and regulates the transport of proteins from the Golgi complex to the cell surface. Later studies have highlighted a role for FAPP2 as lipid transfer protein involved in glycosphingolipid metabolism at the Golgi complex. Here we discuss the available evidence on the function of FAPP2 in both membrane trafficking and lipid metabolism and propose a mechanism of action of FAPP2 that integrates its activities in membrane trafficking and in lipid transfer. This article is part of a Special Issue entitled Lipids and Vesicular Transport.     

Identification of soybean proteins from a single cell type: The root hair

Root hairs (RH) are a terminally differentiated single cell type, mainly involved in water and nutrient uptake from the soil. The soybean RH cell represents an excellent model for the study of single cell systems biology. In this study, we identified 5702 proteins, with at least two peptides, from soybean RH using an accurate mass and time tag approach, establishing a comprehensive proteome reference map of this single cell type. We also showed that trypsin is the most appropriate enzyme for soybean proteomic studies by performing an in silico digestion of the soybean proteome using different proteases. Although the majority of proteins identified in this study are involved in basal metabolism, the function of others are more related to RH formation/function and include proteins involved in nutrient uptake (transporters) or vesicular trafficking (cytoskeleton and ras‐associated binding proteins). Interestingly, some of these proteins appear to be specifically detected in RH and constitute promising candidates for further studies to elucidate unique features of this single‐cell model.     

Evolutionary analysis of the ENTH/ANTH/VHS protein superfamily reveals a coevolution between membrane trafficking and metabolism

ABSTRACT: BACKGROUND: Membrane trafficking involves the complex regulation of proteins and lipids intracellular localization and is required for metabolic uptake, cell growth and development. Different trafficking pathways passing through the endosomes are coordinated by the ENTH/ANTH/VHS adaptor protein superfamily. The endosomes are crucial for eukaryotes since the acquisition of the endomembrane system was a central process in eukaryogenesis. RESULTS: Our in silico analysis of this ENTH/ANTH/VHS superfamily, consisting of proteins gathered from 84 complete genomes representative of the different eukaryotic taxa, revealed that genomic distribution of this superfamily allows to discriminate Fungi and Metazoa from Plantae and Protists. Next, in a four way genome wide comparison, we showed that this discriminative feature is observed not only for other membrane trafficking effectors, but also for proteins involved in metabolism and in cytokinesis, suggesting that metabolism, cytokinesis and intracellular trafficking pathways co-evolved. Moreover, some of the proteins identified were implicated in multiple functions, in either trafficking and metabolism or trafficking and cytokinesis, suggesting that membrane trafficking is central to this co-evolution process. CONCLUSION: Our study suggests that membrane trafficking and compartmentalization were not only key features for the emergence of eukaryotic cells but also drove the separation of the eukaryotes in the different taxa.     

Proteomics on brefeldin A-treated Arabidopsis roots reveals profilin 2 as a new protein involved in the cross-talk between vesicular trafficking and the actin cytoskeleton

The growing importance of vesicular trafficking and cytoskeleton dynamic reorganization during plant development requires the exploitation of novel experimental approaches. Several genetic and cell biological studies have used diverse pharmaceutical drugs that inhibit vesicular trafficking and secretion to study these phenomena. Here, proteomic and cell biology approaches were applied to study effects of brefeldin A (BFA), an inhibitor of vesicle recycling and secretion, in Arabidopsis roots. The main aim of this study was to obtain an overview of proteins affected by BFA, but especially to identify new proteins involved in the vesicular trafficking and its cross-talk to the actin cytoskeleton. The results showed that BFA altered vesicular trafficking and caused the formation of BFA-compartments which was accompanied by differential expression of several proteins in root cells. Some of the BFA-up-regulated proteins belong to the class of the vesicular trafficking proteins, such as V-ATPase and reversibly glycosylated polypeptide, while others, such as profilin 2 and elongation factor 1 alpha, are rather involved in the remodeling of the actin cytoskeleton. Upregulation of profilin 2 by BFA was verified by immunoblot and live imaging at subcellular level. The latter approach also revealed that profilin 2 accumulated in BFA-compartments which was accompanied by remodeling of the actin cytoskeleton in BFA-treated root cells. Thus, profilin 2 seems to be involved in the cross-talk between vesicular trafficking and the actin cytoskeleton, in a BFA-dependent manner.     

Microtubule motors at the intersection of trafficking and transport

Molecular motors drive the transport of vesicles and organelles within the cell. Traditionally, these transport processes have been considered separately from membrane trafficking events, such as regulated budding and fusion. However, recent progress has revealed mechanistic links that integrate these processes within the cell. Rab proteins, which function as key regulators of intracellular trafficking, have now been shown to recruit specific motors to organelle membranes. Rab-independent recruitment of motors by adaptor or scaffolding proteins is also a key mechanism. Once recruited to vesicles and organelles, these motors can then drive directed transport; this directed transport could in turn affect the efficiency of trafficking events. Here, we discuss this coordinated regulation of trafficking and transport, which provides a powerful mechanism for temporal and spatial control of cellular dynamics.     

Trying to make sense of retromer

Retromer is a cytosolic protein complex which binds to post-Golgi organelles involved in the trafficking of proteins to the lytic compartment of the cell. In non-plant organisms, retromer mediates the recycling of acid hydrolase receptors from early endosomal (EE) compartments. In plants, retromer components are required for the targeting of vacuolar storage proteins, and for the recycling of endocytosed PIN proteins. However, there are contradictory reports as to the localization of the sorting nexins and the core subunit of retromer. There is also uncertainty as to the identity of the organelles from which vacuolar sorting receptors (VSRs) and endocytosed plasma membrane (PM) proteins are recycled. In this review we try to resolve some of these conflicting observations.     

GLUT4 trafficking in insulin-sensitive cells

In recent years, there have been major advances in the under-standing of both the cell biology of vesicle trafficking between intracellular compartments and the molecular targeting signals intrinsic to the trafficking proteins themselves. One system to which these advances have been profitably applied is the regulation of the trafficking of a glucose transporter, GLUT4, from intracellular compartment(s) to the cell surface in response to insulin. The unique nature of the trafficking of GLUT4 and its expression in highly differentiated cells makes this a question of considerable interest to cell biologists. Unraveling the tangled web of molecular events coordinating GLUT4 trafficking will eventually lead to a greater understanding of mammalian glucose metabolism, as well as fundamental cell biological principles related to organelle biogenesis and protein trafficking.     

Setting SNAREs in a different wood

Vesicle traffic is essential for cell homeostasis, growth and development in plants, as it is in other eukaryotes, and is facilitated by a superfamily of proteins known as soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors (SNAREs). Although SNAREs are well-conserved across phylla, genomic analysis for two model angiosperm species available to date, rice and Arabidopsis, highlights common patterns of divergence from other eukaryotes. These patterns are associated with the expansion of some gene subfamilies of SNAREs, the absence of others and the appearance of new proteins that show no significant homologies to SNAREs of mammals, yeast or Drosophila. Recent findings indicate that the functions of these plant SNAREs also extend beyond the conventional 'housekeeping' activities associated with vesicle trafficking. A number of SNAREs have been implicated in environmental responses as diverse as stomata movements and gravisensing as well as sensitivity to salt and drought. These proteins are essential for signal transduction and response and, in most cases, appear also to maintain additional roles in membrane trafficking. One common theme to this added functionality lies in control of non-SNARE proteins, notably ion channels. Other examples include interactions between the SNAREs and scaffolding or other structural components within the plant cell.     

Assembly and trafficking of heterotrimeric G proteins

To be activated by cell surface G protein-coupled receptors, heterotrimeric G proteins must localize at the cytoplasmic surface of plasma membranes. Moreover, some G protein subunits are able to traffic reversibly from the plasma membrane to intracellular locations upon activation. This current topic will highlight new insights into how nascent G protein subunits are assembled and how they arrive at plasma membranes. In addition, recent reports have increased our knowledge of activation-induced trafficking of G proteins. Understanding G protein assembly and trafficking will lead to a greater understanding of novel ways that cells regulate G protein signaling.