Membrane traffic in the secretory pathway

Vesicular transport is the basic communication mechanism between different compartments in a cell and with the environment. In this review I discuss the principles of vesicle generation and consumption with particular emphasis on the different types of coat proteins and the timing of the shedding of the coat proteins from transport containers. In recent years it has become clear that there are more coat complexes than the classical COPI, COPII and clathrin coats. These additional coats may generate vesicles that transport cargo in a temporally and/or spatially controlled manner. Work over the last years suggests that GTP hydrolysis occurs early during vesicle biogenesis, destabilizing the coat perhaps before fission of the vesicle from the donor membrane occurs. Recent findings imply, however, that tethers at the receiving compartment specifically detect the coat on vesicle. (Part of a Multi-author Review)     

Membrane traffic in the secretory pathway

Vesicular transport is the basic communication mechanism between different compartments in a cell and with the environment. In this review I discuss the principles of vesicle generation and consumption with particular emphasis on the different types of coat proteins and the timing of the shedding of the coat proteins from transport containers. In recent years it has become clear that there are more coat complexes than the classical COPI, COPII and clathrin coats. These additional coats may generate vesicles that transport cargo in a temporally and/or spatially controlled manner. Work over the last years suggests that GTP hydrolysis occurs early during vesicle biogenesis, destabilizing the coat perhaps before fission of the vesicle from the donor membrane occurs. Recent findings imply, however, that tethers at the receiving compartment specifically detect the coat on vesicle. (Part of a Multi-author Review)     

A role for the hinge/ear domain of the β chains in the incorporation of AP complexes into clathrin-coated pits and coated vesicles

The clathrin-associated adaptor protein (AP) complexes drive the polymerization of clathrin in coated pits to form coated vesicles. It has previously been shown that the carboxyl-terminal hinge/ear domain of the β2 chain contains a binding site for clathrin and that removal of this domain from APs or from isolated β2 chains abrogates their ability to form clathrin coats in vitro. We show here that the hinge/ear domain is necessary for efficient incorporation of AP complexes into coated pits and coated vesicles in cells, a result that is consistent with the view that the β chains indeed provide an important interaction between the AP complexes and clathrin. Received 7 April 1997; received after revision 22 May 1997; accepted 28 May 1997     

Myelin basic protein: a multifunctional protein

Myelin basic protein (MBP), the second most abundant protein in central nervous system myelin, is responsible for adhesion of the cytosolic surfaces of multilayered compact myelin. A member of the ‘intrinsically disordered’ or conformationally adaptable protein family, it also appears to have several other functions. It can interact with a number of polyanionic proteins including actin, tubulin, Ca²⁺-calmodulin, and clathrin, and negatively charged lipids, and acquires structure on binding to them. It may act as a membrane actin-binding protein, which might allow it to participate in transmission of extracellular signals to the cytoskeleton in oligodendrocytes and tight junctions in myelin. Some size isoforms of MBP are transported into the nucleus and thus they may also bind polynucleotides. Extracellular signals received by myelin or cultured oligodendrocytes cause changes in phosphorylation of MBP, suggesting that MBP is also involved in signaling. Further study of this very abundant protein will reveal how it is utilized by the oligodendrocyte and myelin for different purposes. Received 2 March 2006; received after revision 12 April 2006; accepted 16 May 2006     

Molecular machinery mediating vesicle budding,docking and fusion

A general machinery buds and fuses transport vesicles which connect intracellular compartments with each other and allow communication with the extracellular environment. Cytoplasmic coat proteins deform membranes to bud vesicles and interact directly or indirectly with cargo molecules. Compartment-specific SNAREs (SNAP receptors) on vesicles and target membranes dock vesicles and provide a scaffolding for the general fusion machinery to initiate lipid bilayer fusion.     

HIP1 exhibits an early recruitment and a late stage function in the maturation of coated pits

Huntingtin interacting protein 1 (HIP1) is an accessory protein of the clathrin-mediated endocytosis (CME) pathway, yet its precise role and the step at which it becomes involved are unclear. We employed live-cell imaging techniques to focus on the early steps of CME and characterize HIP1 dynamics. We show that HIP1 is highly colocalized with clathrin at the plasma membrane and shares similar dynamics with a subpopulation of clathrin assemblies. Employing transferrin receptor fused to pHluorin, we distinguished between open pits to which HIP1 localizes and newly internalized vesicles that are devoid of HIP1. Moreover, shRNA knockdown of clathrin compromised HIP1 membranal localization, unlike the reported behavior of Sla2p. HIP1 fragment, lacking its ANTH and Talin-like domains, inhibits internalization of transferrin, but retains colocalization with membranal clathrin assemblies. These data demonstrate HIP1’s role in pits maturation and formation of the coated vesicle, and its strong dependence on clathrin for membranal localization. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Trans-Golgi network sorting

The trans-Golgi network (TGN) is a major secretory pathway sorting station that directs newly synthesized proteins to different subcellular destinations. The TGN also receives extracellular materials and recycled molecules from endocytic compartments. In this review, we summarize recent progress on understanding TGN structure and the dynamics of trafficking to and from this compartment. Protein sorting into different transport vesicles requires specific interactions between sorting motifs on the cargo molecules and vesicle coat components that recognize these motifs. Current understanding of the various targeting signals and vesicle coat components that are involved in TGN sorting are discussed, as well as the molecules that participate in retrieval to this compartment in both yeast and mammalian cells. Besides proteins, lipids and lipid-modifying enzymes also participate actively in the formation of secretory vesicles. The possible mechanisms of action of these lipid hydrolases and lipid kinases are discussed. Finally, we summarize the fundamentally different apical and basolateral cell surface delivery mechanisms and the current facts and hypotheses on protein sorting from the TGN into the regulated secretory pathway in neuroendocrine cells. Received 2 November 2000; received after revision 19 February 2001; accepted 19 February 2001     

The structural and mechanistic basis for recycling of Rab proteins between membrane compartments

Rab proteins are members of the Ras superfamily of GTPases and are key regulators of intracellular vesicular transport. They undergo a cycle of GTPase activity, and this activity is interconnected to a cycle of reversible attachment to membranes. This cycle is mediated by geranylgeranylation of (usually) two C-terminal cysteines, which in turn is effected by Rab geranylgeranyltransferase in concert with REP (Rab escort protein). After delivery to their respective membranes, Rabs are activated by replacement of GDP by GTP, allowing interaction with a wide variety of effector molecules involved in vesicular transport, in particular with docking of transport vesicles to their specific target membranes. After completion of these events and GTP hydrolysis, Rabs are retrieved by GDI (GDP dissociation inhibitor) and delivered to their starting compartment. Here, the structural and mechanistic basis of events occurring in Rab delivery and cycling, and the differences between REP and GDI are discussed on the basis of recent advances in the field.Received 4 November 2004; received after revision 14 February 2005; accepted 31 March 2005     

New insights into the molecular mechanisms of sperm-egg interaction

At the moment of insemination millions of mammalian sperm cells are released into the female reproductive tract in order to find a single cell – the oocyte. The spermatozoa subsequently ignore the thousands of cells they make contact with during their journey to the site of fertilisation, until they reach the surface of the oocyte. At this point, they bind tenaciously to the acellular coat, known as the zona pellucida, that surrounds the oocyte and initiate the chain of cellular interactions that will culminate in fertilization. These exquisitely cell- and species-specific recognition events are among the most strategically important cellular interactions in biology. Understanding the cellular and molecular mechanisms that underpin them has implications for diagnosis of the aetiology of human infertility and the development of novel targets for fertility regulation. Herein, we describe two models indicating the plethora of highly orchestrated molecular interactions underlying successful sperm zona binding and sperm oocyte fusion. Received 17 December 2006; received after revision 31 January 2007; accepted 16 March 2007     

Dictyostelium discoideum cells shed vesicles with associated DNA and vital stain Hoechst 33342

Dictyostelium discoideum cells are highly resis tant to xenobiotics. We previously observed that these primitive eukaryotic cells contain a 170-kDa P-glycoprotein, mediating multidrug resistance in mammalian cells, but nonfunctional in Dictyostelium cells. We show here that D. discoideum cells vitally stained with the DNA-specific dye, Hoechst 33342, release fluorescent material in their culture medium. Electron microscopy and lipid analysis demonstrate the vesicular nature of this material. Moreover, nucleic acids associate with these extracellular vesicles independently of Hoechst vital staining. The main vesicular DNA component exhibits a size >21 kb. Shedding of microvesicles during cell growth is not concomitant with programmed cell death. We propose that these extracellular vesicles are involved in a new cellular resistance mechanism against xenobiotics. Furthermore, since the association of DNA with vesicles occurs in physiological growth conditions and independently of vital staining, the new shedding process might be involved in a more general intercellular mechanism. Received 14 November 1997; received after revision 16 March 1998; accepted 16 March 1998     

Farnesylation of Pex19p is not essential for peroxisome biogenesis in yeast and mammalian cells

Pex19p exhibits a broad binding specificity for peroxisomal membrane proteins (PMPs), and is essential for the formation of functional peroxisomal membranes. Pex19p orthologues contain a C-terminal CAAX motif common to prenylated proteins. In addition, Saccharomyces cerevisiae and Chinese hamster Pex19p are at least partially farnesylated in vivo. Whether farnesylation of Pex19p plays an essential or merely ancillary role in peroxisome biogenesis is currently not clear. Here, we show that (i) nonfarnesylated and farnesylated human Pex19p display a similar affinity towards a select set of PMPs, (ii) a variant of Pex19p lacking a functional farnesylation motif is able to restore peroxisome biogenesis in Pex19p-deficient cells, and (iii) peroxisome protein import is not affected in yeast and mammalian cells defective in one of the enzymes involved in the farnesylation pathway. Summarized, these observations indicate that the CAAX box-mediated processing steps of Pex19p are dispensable for peroxisome biogenesis in yeast and mammalian cells. Received 10 March 2006; received after revision 28 April 2006; accepted 30 May 2006     

Low-density lipoprotein receptor-mediated endocytosis of PEGylated nanoparticles in rat brain endothelial cells

Poly(methoxypolyethyleneglycol cyanoacrylate-co-hexadecylcyanoacrylate) (PEG-PHDCA) nanoparticles have demonstrated their capacity to diffuse through the blood-brain barrier after intravenous administration. However, the mechanism of transport of these nanoparticles into brain has not yet been clearly elucidated. The development of a model of rat brain endothelial cells (RBEC) in culture has allowed investigations into this mechanism. A study of the intracellular trafficking of nanoparticles by cell fractionation and confocal microscopy showed that nanoparticles are internalized by the endocytic pathway. Inhibition of the caveolae-mediated pathway by preincubation with filipin and nystatin did not modify the cellular uptake of the nanoparticles. In contrast, chlorpromazine and NaN₃ pretreatment, which interferes with clathrin and energy-dependent endocytosis, caused a significant decrease of nanoparticle internalization. Furthermore, cellular uptake experiments with nanoparticles preincubated with apolipoprotein E and blocking of low-density lipoprotein receptors (LDLR) clearly suggested that the LDLR-mediated pathway was involved in the endocytosis of PEGPHDCA nanoparticles by RBEC. Received 1 September 2006; received after revision 4 December 2006; accepted 18 December 2006     

Expansion of amino acid homo-sequences in proteins: Insights into the role of amino acid homo-polymers and of the protein context in aggregation

Expansion of amino acid homo-sequences, such as polyglutamines or polyalanines, in proteins has been directly implicated in various degenerative diseases through a mechanism of protein misfolding and aggregation. However, it is still unclear how the nature of the expansion and the protein context influence the tendency of a protein to aggregate. Here, we have addressed these questions using spinocerebellar ataxia type-3 (ATX3) protein, the best characterised of the polyglutamine proteins, chosen as a model system. Using a transfected mammalian cell line, we demonstrate that ATX3 aggregation is noticeably reduced by deletion or replacement of regions other than the polyglutamine tract. The nature of the amino acid homo-sequences also has a strong influence on aggregation. From our studies, we draw general conclusions on the effect of the protein architecture and of the amino acid homo-sequence on pathology. Received 3 March 2006; received after revision 19 April 2006; accepted 22 May 2006     

Lipid transport in microorganisms

Summary Microorganisms are useful model systems for the study of intracellular transport of lipids. Eukaryotic microorganisms, such as the yeastSaccharomyces cerevisiae, are similar to higher eukaryotes with respect to organelle structure and membrane assembly. Experiments in vivo showed that transport of phosphatidylcholine between yeast microsomes and mitochondria is energy independent; transfer of phosphatidylinositol to the plasma membrane and the flux of secretory vesicles take place by different mechanisms. Linkage of transfer and biosynthesis of phospholipids was demonstrated in the case of intramitochondrial phospholipid transfer. A yeast phosphatidylinositol/phosphatidylcholine transfer protein, which is essential for cell viability, was isolated and characterized. Another phospholipid transfer protein present in yeast cytosol, which has a different specificity, is currently under investigation. Transfer of phospholipids between cellular membranes was also demonstrated with prokaryotes. The cytoplasm and the periplasma of the gram-negative facultative photosynthetic bacteriumRhodopseudomonas sphaeroides contain phospholipid transfer proteins; these seem to be involved in the biosynthesis of prokaryotic membranes.     

Proteomic analysis of low-abundant integral plasma membrane proteins based on gels

To characterize low-copy integral membrane proteins and offer some methods for human liver proteome projects, we fractionated highly purified rat liver plasma membrane (PM). PM was purified through two sucrose density gradient centrifugations, and treated with 0.1 M Na₂CO₃, chloroform/methanol and Triton X-100. Proteins were separated by electrophoresis and submitted to mass spectrometry analysis. Four hundred and fiftyseven non-redundant membrane proteins were identified, of which 23% (105) were integral membrane proteins with one or more transmembrane domains. One hundred and fifty-three (33.5%) had no location annotation and 68 were unknown-function proteins. The proteins from different fractions were complementory. A database search for all identified proteins revealed that 53 proteins were involved in the cell communication pathway. More interestingly, more than 50% of the proteins had a protein abundance index concentration of less than 0.1 mol/l, and 12% proteins a concentration 100 times less than that of arginase 1 and actin. Received 15 March 2006; received after revision 17 May 2006; accepted 10 June 2006 L.-J. Zhang and X.-e Wang are contributed equally to this work.     

Membrane-shed vesicles from the parasite <Emphasis Type="Italic">Trichomonas vaginalis</Emphasis>: characterization and their association with cell interaction

Trichomonas vaginalis is a common sexually transmitted parasite that colonizes the human urogenital tract, where it remains extracellular and adheres to epithelial cells. Infections range from asymptomatic to highly inflammatory, depending on the host and the parasite strain. Despite the serious consequences associated with trichomoniasis disease, little is known about parasite or host factors involved in attachment of the parasite-to-host epithelial cells. Here, we report the identification of microvesicle-like structures (MVs) released by T. vaginalis. MVs are considered universal transport vehicles for intercellular communication as they can incorporate peptides, proteins, lipids, miRNA, and mRNA, all of which can be transferred to target cells through receptor–ligand interactions, fusion with the cell membrane, and delivery of a functional cargo to the cytoplasm of the target cell. In the present study, we demonstrated that T. vaginalis release MVs from the plasma and the flagellar membranes of the parasite. We performed proteomic profiling of these structures demonstrating that they possess physical characteristics similar to mammalian extracellular vesicles and might be selectively charged with specific protein content. In addition, we demonstrated that viable T. vaginalis parasites release large vesicles (LVs), membrane structures larger than 1 µm that are able to interact with other parasites and with the host cell. Finally, we show that both populations of vesicles present on the surface of T vaginalis are induced in the presence of host cells, consistent with a role in modulating cell interactions.     

Annexin V interactions with collagen

Annexin V was originally identified as a collagen-binding protein called anchorin CII and was isolated from chondrocyte membranes by affinity chromatography on native type II collagen. The binding of annexin V to native collagen type II is stable at physiological ionic strength when annexin V is reconstituted in liposomes. The binding to native collagen types II and X, and to some extent to type I as well, was confirmed using recombinant annexin V. A physiological role for annexin V interactions with extracellular collagen is consistent with the localization of annexin V on the outer cell surface of chondrocytes, microvilli of hypertrophic chondrocytes, fibroblasts and osteoblasts. A breakthrough in our understanding of the function of annexin V was made with the discovery of its calcium channel activity. At least one of several putative functions of annexin V became obvious from studies on matrix vesicles derived from calcifying cartilage. It was found that calcium uptake by matrix vesicles depend on collagen type II and type X binding to annexin V in the vesicles and was lost when collagens were digested with collagenase; calcium influx was reconstituted after adding back native collagen II or V. These findings indicate that annexin V plays a major role in matrix vesicle-initiated cartilage calcification as a collagen-regulated calcium channel.     

Ribosome-inactivating proteins: progress and problems

Ribosome-inactivating proteins (RIPs), mostly from plants, are enzymes which depurinate rRNA, thus inhibiting protein synthesis. They also depurinate other polynucleotide substrates. The biological activity of RIPs is not completely clarified, and sometimes independent of the inhibition of protein synthesis. There are differences in the cytotoxicity of RIPs and, consequently, in their toxicity to animals. Some RIPs are potent toxins, the best known being ricin, a potential biological weapon. New toxins have recently been identified. RIPs cause apoptotic and necrotic lesions, and induce production of cytokines causing inflammation. RIPs are potentially useful in agriculture and medicine because (i) they have antiviral activity and (ii) they are used for the preparation of conjugates with antibodies (‘immunotoxins’) or other carriers, rendering them specifically toxic to the cell target of the carrier, which may be helpful in therapy. The distribution, mechanism of action and role in nature of RIPs are not completely understood, and we can expect several future developments in their practical application. Received 17 February 2006; received after revision 23 March 2006; accepted 2 May 2006     

Functions of actin in endocytosis

Endocytosis is a fundamental eukaryotic process required for remodelling plasma-membrane lipids and protein to ensure appropriate membrane composition. Increasing evidence from a number of cell types reveals that actin plays an active, and often essential, role at key endocytic stages. Much of our current mechanistic understanding of the endocytic process has come from studies in budding yeast and has been facilitated by yeast’s genetic amenability and by technological advances in live cell imaging. While endocytosis in metazoans is likely to be subject to a greater array of regulatory signals, recent reports indicate that spatiotemporal aspects of vesicle formation requiring actin are likely to be conserved across eukaryotic evolution. In this review we focus on the ‘modular’ model of endocytosis in yeast before highlighting comparisons with other cell types. Our discussion is limited to endocytosis involving clathrin as other types of endocytosis have not been demonstrated in yeast.     

Molecular aspects of membrane fission in the secretory pathway

Membrane fission is essential in various intracellular dissociative transport steps. The molecular mechanisms by which endocytic vesicles detach from the plasma membrane are being rapidly elucidated. Much less is known about the fission mechanisms operating at Golgi tubular networks; these include the Golgi transport and sorting stations, the trans-Golgi and cis-Golgi networks, where the geometry and physical properties of the membranes differ from those at the cell surface. Here we discuss the lipid and protein machineries that have so far been related to the fission process, with emphasis on those acting in the Golgi complex. Received 10 May 2002; received after revision 20 June 2002; accepted 26 June 2002 RID="*" ID="*"Corresponding author.