Comparative proximity biotinylation implicates the small GTPase RAB18 in sterol mobilization and biosynthesis.

Loss of functional RAB18 causes the autosomal recessive condition Warburg Micro syndrome. To better understand this disease, we used proximity biotinylation to generate an inventory of potential RAB18 effectors. A restricted set of 28 RAB18 interactions were dependent on the binary RAB3GAP1-RAB3GAP2 RAB18-guanine nucleotide exchange factor complex. Twelve of these 28 interactions are supported by prior reports, and we have directly validated novel interactions with SEC22A, TMCO4, and INPP5B. Consistent with a role for RAB18 in regulating membrane contact sites, interactors included groups of microtubule/membrane-remodeling proteins, membrane-tethering and docking proteins, and lipid-modifying/transporting proteins. Two of the putative interactors, EBP and OSBPL2/ORP2, have sterol substrates. EBP is a Δ8-Δ7 sterol isomerase, and ORP2 is a lipid transport protein. This prompted us to investigate a role for RAB18 in cholesterol biosynthesis. We found that the cholesterol precursor and EBP-product lathosterol accumulates in both RAB18-null HeLa cells and RAB3GAP1-null fibroblasts derived from an affected individual. Furthermore, de novo cholesterol biosynthesis is impaired in cells in which RAB18 is absent or dysregulated or in which ORP2 expression is disrupted. Our data demonstrate that guanine nucleotide exchange factor-dependent Rab interactions are highly amenable to interrogation by proximity biotinylation and may suggest that Micro syndrome is a cholesterol biosynthesis disorder.

Adaptation of Leishmania donovani to cutaneous and visceral environments: in vivo selection and proteomic analysis.

Leishmaniasis is a neglected tropical disease caused by Leishmania protozoa. Two main forms are found in the Old World, self-limited cutaneous leishmaniasis and potentially fatal visceral leishmaniasis, with parasite dissemination to liver, bone marrow, and spleen. The Leishmania donovani species complex is the causative agent of visceral leishmaniasis worldwide, but atypical L. donovani strains can cause cutaneous leishmaniasis. We hypothesized that L. donovani can adapt to survive in response to restrictions imposed by the host environment. To assess this, we performed in vivo selection in BALB/c mice with a cutaneous L. donovani clinical isolate to select for parasites with increased capacity to survive in visceral organs. We then performed whole cell proteomic analysis and compared this visceral-selected strain to the original cutaneous clinical isolate and to a visceral leishmaniasis clinical isolate. Overall, there were no major shifts in proteomic profiles; however, translation, biosynthetic processes, antioxidant protection, and signaling were elevated in visceral strains. Conversely, transport and trafficking were elevated in the cutaneous strain. Overall, these results provide new insight into the adaptability of Leishmania parasites to the host environment and on the factors that mediate their ability to survive in different organs.

Quantitative proteomics reveals differentially expressed proteins in murine preneoplastic intestine in a model of intestinal tumorigenesis induced by low dietary folate and MTHFR deficiency.

Colorectal cancer risk is increased when dietary folate intake is low, with or without a deficiency in methylenetetrahydrofolate reductase (MTHFR). We have observed that intestinal tumors are induced in mice fed low-folate diets, and that tumor incidence is increased when these mice also have MTHFR deficiency. This study was undertaken to identify differentially expressed proteins in conditions favoring initial steps of murine carcinogenesis in normal preneoplastic intestine. We compared the proteome of BALB/c normal intestine in Mthfr(+/+) mice fed control diets for 1 year (low susceptibility to tumorigenesis) with the proteome of Mthfr(+/-) animals fed low folate diets (higher tumor susceptibility). Our data suggest that the NuRD complex, KRAS-related proteins, the protein synthetic machinery, and fatty acid-related metabolic proteins are upregulated in the early stages of tumorigenesis. These proteins may serve as biomarkers or targets for colorectal cancer diagnosis or therapy.

Organellar proteomics to create the cell map.

The elucidation of a complete, accurate, and permanent representation of the proteome of the mammalian cell may be achievable piecemeal by an organellar based approach. The small volume of organelles assures high protein concentrations. Providing isolated organelles are homogenous, this assures reliable protein characterization within the sensitivity and dynamic range limits of current mass spec based analysis. The stochastic aspect of peptide selection by tandem mass spectrometry for sequence determination by fragmentation is dealt with by multiple biological replicates as well as by prior protein separation on 1-D gels. Applications of this methodology to isolated synaptic vesicles, clathrin coated vesicles, endosomes, phagosomes, endoplasmic reticulum, and Golgi apparatus, as well as Golgi-derived COPI vesicles, have led to mechanistic insight into the identity and function of these organelles.

SNARE proteins mediate fusion between cytosolic lipid droplets and are implicated in insulin sensitivity.

The accumulation of cytosolic lipid droplets in muscle and liver cells has been linked to the development of insulin resistance and type 2 diabetes. Such droplets are formed as small structures that increase in size through fusion, a process that is dependent on intact microtubules and the motor protein dynein. Approximately 15% of all droplets are involved in fusion processes at a given time. Here, we show that lipid droplets are associated with proteins involved in fusion processes in the cell: NSF (N-ethylmaleimide-sensitive-factor), alpha-SNAP (soluble NSF attachment protein) and the SNAREs (SNAP receptors), SNAP23 (synaptosomal-associated protein of 23 kDa), syntaxin-5 and VAMP4 (vesicle-associated membrane protein 4). Knockdown of the genes for SNAP23, syntaxin-5 or VAMP4, or microinjection of a dominant-negative mutant of alpha-SNAP, decreases the rate of fusion and the size of the lipid droplets. Thus, the SNARE system seems to have an important role in lipid droplet fusion. We also show that oleic acid treatment decreases the insulin sensitivity of heart muscle cells, and this sensitivity is completely restored by transfection with SNAP23. Thus, SNAP23 might be a link between insulin sensitivity and the inflow of fatty acids to the cell.

An implementation framework for the feedback of individual research results and incidental findings in research.

BACKGROUND: This article outlines procedures for the feedback of individual research data to participants. This feedback framework was developed in the context of a personalized medicine research project in Canada. Researchers in this domain have an ethical obligation to return individual research results and/or material incidental findings that are clinically significant, valid and actionable to participants. Communication of individual research data must proceed in an ethical and efficient manner. Feedback involves three procedural steps: assessing the health relevance of a finding, re-identifying the affected participant, and communicating the finding. Re-identification requires researchers to break the code in place to protect participant identities. Coding systems replace personal identifiers with a numerical code. Double coding systems provide added privacy protection by separating research data from personal identifying data with a third "linkage" database. A trusted and independent intermediary, the "keyholder", controls access to this linkage database. DISCUSSION: Procedural guidelines for the return of individual research results and incidental findings are lacking. This article outlines a procedural framework for the three steps of feedback: assessment, re-identification, and communication. This framework clarifies the roles of the researcher, Research Ethics Board, and keyholder in the process. The framework also addresses challenges posed by coding systems. Breaking the code involves privacy risks and should only be carried out in clearly defined circumstances. Where a double coding system is used, the keyholder plays an important role in balancing the benefits of individual feedback with the privacy risks of re-identification. Feedback policies should explicitly outline procedures for the assessment of findings, and the re-identification and contact of participants. The responsibilities of researchers, the Research Ethics Board, and the keyholder must be clearly defined. We provide general guidelines for keyholders involved in feedback. We also recommend that Research Ethics Boards should not be directly involved in the assessment of individual findings. Hospitals should instead establish formal, interdisciplinary clinical advisory committees to help researchers determine whether or not an uncertain finding should be returned.

Mass spectrometry in high-throughput proteomics: ready for the big time.

Mass spectrometry has evolved and matured to a level where it is able to assess the complexity of the human proteome. We discuss some of the expected challenges ahead and promising strategies for success.

A HUPO test sample study reveals common problems in mass spectrometry-based proteomics.

We performed a test sample study to try to identify errors leading to irreproducibility, including incompleteness of peptide sampling, in liquid chromatography-mass spectrometry-based proteomics. We distributed an equimolar test sample, comprising 20 highly purified recombinant human proteins, to 27 laboratories. Each protein contained one or more unique tryptic peptides of 1,250 Da to test for ion selection and sampling in the mass spectrometer. Of the 27 labs, members of only 7 labs initially reported all 20 proteins correctly, and members of only 1 lab reported all tryptic peptides of 1,250 Da. Centralized analysis of the raw data, however, revealed that all 20 proteins and most of the 1,250 Da peptides had been detected in all 27 labs. Our centralized analysis determined missed identifications (false negatives), environmental contamination, database matching and curation of protein identifications as sources of problems. Improved search engines and databases are needed for mass spectrometry-based proteomics.

ARFGAP2 and ARFGAP3 are essential for COPI coat assembly on the Golgi membrane of living cells.

Coat protein complex I (COPI) vesicles play a central role in the recycling of proteins in the early secretory pathway and transport of proteins within the Golgi stack. Vesicle formation is initiated by the exchange of GDP for GTP on ARF1 (ADP-ribosylation factor 1), which, in turn, recruits the coat protein coatomer to the membrane for selection of cargo and membrane deformation. ARFGAP1 (ARF1 GTPase-activating protein 1) regulates the dynamic cycling of ARF1 on the membrane that results in both cargo concentration and uncoating for the generation of a fusion-competent vesicle. Two human orthologues of the yeast ARFGAP Glo3p, termed ARFGAP2 and ARFGAP3, have been demonstrated to be present on COPI vesicles generated in vitro in the presence of guanosine 5'-3-O-(thio)triphosphate. Here, we investigate the function of these two proteins in living cells and compare it with that of ARFGAP1. We find that ARFGAP2 and ARFGAP3 follow the dynamic behavior of coatomer upon stimulation of vesicle budding in vivo more closely than does ARFGAP1. Electron microscopy of ARFGAP2 and ARFGAP3 knockdowns indicated Golgi unstacking and cisternal shortening similarly to conditions where vesicle uncoating was blocked. Furthermore, the knockdown of both ARFGAP2 and ARFGAP3 prevents proper assembly of the COPI coat lattice for which ARFGAP1 does not seem to play a major role. This suggests that ARFGAP2 and ARFGAP3 are key components of the COPI coat lattice and are necessary for proper vesicle formation.

Biogenesis of lipid droplets--how cells get fatter.

Lipid droplets are discrete organelles present in most cell types and organisms including bacteria, yeast, plants, insects and animals. Long considered as passive storage deposits, recent cell biology, proteomic and lipidomic analysis show that lipid droplets are dynamic organelles involved in multiple cellular functions. They have a central function in lipid distribution to different membrane-bound organelles and serve not only as main reservoirs of neutral lipids such as triglycerides and cholesterol but in addition, contain structural proteins, proteins involved in lipid synthesis and transmembrane proteins. A detailed model for how transmembrane proteins such as SNARE proteins can exist in lipid droplets is proposed.

In a mirror dimly: tracing the movements of molecules in living cells.

The random movement of molecules (diffusion) is fundamental to most cellular processes, including enzymatic reactions, signalling, protein-protein interaction, as well as domain and pattern formation. Despite playing a central role, diffusion is, to a large extent, under-appreciated in the cell biology community. One reason for this is that diffusion is rather challenging to study in living cells. This article is intended to explain, at least in part, how we can go about studying diffusion of molecules in living cells, why it is important and how it provides us with important clues about biological systems. As the title 'In a mirror dimly' suggests, we do this by monitoring faint light emitted by fluorescent probes or proteins using advanced optics (e.g. mirrors) and electronics. The data are then fitted and interpreted with mathematical and physical models, providing a glimpse into the world of molecules.

Biogenesis of tubular ER-to-Golgi transport intermediates.

Tubular transport intermediates (TTIs) have been described as one class of transport carriers in endoplasmic reticulum (ER)-to-Golgi transport. In contrast to vesicle budding and fusion, little is known about the molecular regulation of TTI synthesis, transport and fusion with target membranes. Here we have used in vivo imaging of various kinds of GFP-tagged proteins to start to address these questions. We demonstrate that under steady-state conditions TTIs represent approximately 20% of all moving transport carriers. They increase in number and length when more transport cargo becomes available at the donor membrane, which we induced by either temperature-related transport blocks or increased expression of the respective GFP-tagged transport markers. The formation and motility of TTIs is strongly dependent on the presence of intact microtubules. Microinjection of GTPgammaS increases the frequency of TTI synthesis and the length of these carriers. When Rab proteins are removed from membranes by microinjection of recombinant Rab-GDI, the synthesis of TTIs is completely blocked. Microinjection of the cytoplasmic tails of the p23 and p24 membrane proteins also abolishes formation of p24-containing TTIs. Our data suggest that TTIs are ER-to-Golgi transport intermediates that form preferentially when transport-competent cargo exists in excess at the donor membrane. We propose a model where the interaction of the cytoplasmic tails of membrane proteins with microtubules are key determinants for TTI synthesis and may also serve as a so far unappreciated model for aspects of transport carrier formation.

Regulation of microtubule-dependent recycling at the trans-Golgi network by Rab6A and Rab6A'.

The small GTPase rab6A but not the isoform rab6A' has previously been identified as a regulator of the COPI-independent recycling route that carries Golgi-resident proteins and certain toxins from the Golgi to the endoplasmic reticulum (ER). The isoform rab6A' has been implicated in Golgi-to-endosomal recycling. Because rab6A but not A', binds rabkinesin6, this motor protein is proposed to mediate COPI-independent recycling. We show here that both rab6A and rab6A' GTP-restricted mutants promote, with similar efficiency, a microtubule-dependent recycling of Golgi resident glycosylation enzymes upon overexpression. Moreover, we used small interfering RNA mediated down-regulation of rab6A and A' expression and found that reduced levels of rab6 perturbs organization of the Golgi apparatus and delays Golgi-to-ER recycling. Rab6-directed Golgi-to-ER recycling seems to require functional dynactin, as overexpression of p50/dynamitin, or a C-terminal fragment of Bicaudal-D, both known to interact with dynactin inhibit recycling. We further present evidence that rab6-mediated recycling seems to be initiated from the trans-Golgi network. Together, this suggests that a recycling pathway operates at the level of the trans-Golgi linking directly to the ER. This pathway would be the preferred route for both toxins and resident Golgi proteins.

Non-alcoholic fatty liver disease--a chronic disease of the 21st century.

Non-alcoholic fatty liver disease (NAFLD) comprises a spectrum of metabolic states ranging from simple steatosis to inflammation with associated fibrosis to cirrhosis. Though accumulation of hepatic fat is not associated with a significant increase in mortality rates, hepatic inflammation is, as this augments the risk of terminal liver disease, i.e., cirrhosis, hepatic decompensation (liver failure) and/or hepatocellular carcinoma. Disease progression is usually slow, over a decade or more and, for the most part, remains asymptomatic. Recent estimates suggest that the global prevalence of NAFLD is high, about one in four. In most cases, NAFLD overlaps with overweight, obesity, cardiovascular disease and the metabolic syndrome with numerous contributing parameters including a dysregulation of adipose tissue, insulin resistance, type 2 diabetes, changes in the gut microbiome, neuronal and hormonal dysregulation and metabolic stress. NAFLD is diagnosed incidentally, despite its high prevalence. Non-invasive imaging techniques have emerged, making it possible to determine degree of steatosis as well asfibrosis. Despite this, the benefit of routine diagnostics remains uncertain. A better understanding of the (molecular) pathogenesis of NAFLD is needed combined with long-term studies where benefits of treatment can be assessed to determine cost-benefit ratios. This review summarizes the current state of knowledge and possible areas of treatment.

Association of the Golgi UDP-galactose transporter with UDP-galactose:ceramide galactosyltransferase allows UDP-galactose import in the endoplasmic reticulum.

UDP-galactose reaches the Golgi lumen through the UDP-galactose transporter (UGT) and is used for the galactosylation of proteins and lipids. Ceramides and diglycerides are galactosylated within the endoplasmic reticulum by the UDP-galactose:ceramide galactosyltransferase. It is not known how UDP-galactose is transported from the cytosol into the endoplasmic reticulum. We transfected ceramide galactosyltransferase cDNA into CHOlec8 cells, which have a defective UGT and no endogenous ceramide galactosyltransferase. Cotransfection with the human UGT1 greatly stimulated synthesis of lactosylceramide in the Golgi and of galactosylceramide in the endoplasmic reticulum. UDP-galactose was directly imported into the endoplasmic reticulum because transfection with UGT significantly enhanced synthesis of galactosylceramide in endoplasmic reticulum membranes. Subcellular fractionation and double label immunofluorescence microscopy showed that a sizeable fraction of ectopically expressed UGT and ceramide galactosyltransferase resided in the endoplasmic reticulum of CHOlec8 cells. The same was observed when UGT was expressed in human intestinal cells that have an endogenous ceramide galactosyltransferase. In contrast, in CHOlec8 singly transfected with UGT 1, the transporter localized exclusively to the Golgi complex. UGT and ceramide galactosyltransferase were entirely detergent soluble and form a complex because they could be coimmunoprecipitated. We conclude that the ceramide galactosyltransferase ensures a supply of UDP-galactose in the endoplasmic reticulum lumen by retaining UGT in a molecular complex.

Commuting between Golgi cisternae--mind the GAP!

Intracellular transport has remained central to cell biology now for more than 40 years. Despite this, we still lack an overall mechanistic framework that describes transport in different parts of the cell. In the secretory pathway, basic questions, such as how biosynthetic cargo traverses the pathway, are still debated. Historically, emphasis was first put on interpreting function from morphology at the ultrastructural level revealing membrane structures such as the transitional ER, vesicular carriers, vesicular tubular clusters, Golgi cisternae, Golgi stacks and the Golgi ribbon. This emphasis on morphology later switched to biochemistry and yeast genetics yielding many of the key molecular players and their associated functions that we know today. More recently, microscopy studies of living cells incorporating biophysics and system analysis has proven useful and is often used to readdress earlier findings, sometimes with surprising outcomes.

The TRAPP Subunit Trs130p Interacts with the GAP Gyp6p to Mediate Ypt6p Dynamics at the Late Golgi.

Small GTPases of the Rab superfamily participate in virtually all vesicle-mediated trafficking events. Cycling between an active GTP-bound form and an inactive GDP-bound form is accomplished in conjunction with guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs), respectively. Rab cascades have been described in which an effector of an activated Rab is a GEF for a downstream Rab, thus ensuring activation of a pathway in an ordered fashion. Much less is known concerning crosstalk between GEFs and GAPs although regulation between these factors could also contribute to the overall physiology of a cell. Here we demonstrate that a subunit of the TRAPP II multisubunit tethering factor, a Rab GEF, participates in the recruitment of Gyp6p, a GAP for the GTPase Ypt6p, to Golgi membranes. The extreme carboxy-terminal portion of the TRAPP II subunit Trs130p is required for the interaction between TRAPP II and Gyp6p. We further demonstrate that TRAPP II mutants, but not a TRAPP III mutant, display a defect in Gyp6p interaction. A consequence of this defective interaction is the enhanced localization of Ypt6p at late Golgi membranes. Although a ypt31/32 mutant also resulted in an enhanced localization of Gyp6p at the late Golgi, the effect was not as dramatic as that seen for TRAPP II mutants, nor was Ypt31/32 detected in the same TRAPP II purification that detected Gyp6p. We propose that the interaction between TRAPP II and Gyp6p represents a parallel mechanism in addition to that mediated by Ypt31/32 for the recruitment of a GAP to the appropriate membrane, and is a novel example of crosstalk between a Rab GAP and GEF.

Compartmentalization of membrane trafficking, glucose transport, glycolysis, actin, tubulin and the proteasome in the cytoplasmic droplet/Hermes body of epididymal sperm.

Discovered in 1909 by Retzius and described mainly by morphology, the cytoplasmic droplet of sperm (renamed here the Hermes body) is conserved among all mammalian species but largely undefined at the molecular level. Tandem mass spectrometry of the isolated Hermes body from rat epididymal sperm characterized 1511 proteins, 43 of which were localized to the structure in situ by light microscopy and two by quantitative electron microscopy localization. Glucose transporter 3 (GLUT-3) glycolytic enzymes, selected membrane traffic and cytoskeletal proteins were highly abundant and concentrated in the Hermes body. By electron microscope gold antibody labelling, the Golgi trafficking protein TMED7/p27 localized to unstacked flattened cisternae of the Hermes body, as did GLUT-3, the most abundant protein. Its biogenesis was deduced through the mapping of protein expression for all 43 proteins during male germ cell differentiation in the testis. It is at the terminal step 19 of spermiogenesis that the 43 characteristic proteins accumulated in the nascent Hermes body.

Expression, sorting, and segregation of Golgi proteins during germ cell differentiation in the testis.

The molecular basis of changes in structure, cellular location, and function of the Golgi apparatus during male germ cell differentiation is unknown. To deduce cognate Golgi proteins, we isolated germ cell Golgi fractions, and 1318 proteins were characterized, with 20 localized in situ. The most abundant protein, GL54D of unknown function, is characterized as a germ cell-specific Golgi-localized type II integral membrane glycoprotein. TM9SF3, also of unknown function, was revealed to be a universal Golgi marker for both somatic and germ cells. During acrosome formation, several Golgi proteins (GBF1, GPP34, GRASP55) localize to both the acrosome and Golgi, while GL54D, TM9SF3, and the Golgi trafficking protein TMED7/p27 are segregated from the acrosome. After acrosome formation, GL54D, TM9SF3, TMED4/p25, and TMED7/p27 continue to mark Golgi identity as it migrates away from the acrosome, while the others (GBF1, GPP34, GRASP55) remain in the acrosome and are progressively lost in later steps of differentiation. Cytoplasmic HSP70.2 and the endoplasmic reticulum luminal protein-folding enzyme PDILT are also Golgi recruited but only during acrosome formation. This resource identifies abundant Golgi proteins that are expressed differentially during mitosis, meiosis, and postacrosome Golgi migration, including the last step of differentiation.

Rab proteins implicated in lipid storage and mobilization.

Abnormal intracellular accumulation or transport of lipids contributes greatly to the pathogenesis of human diseases. In the liver, excess accumulation of triacylglycerol (TG) leads to fatty liver disease encompassing steatosis, steatohepatitis and fibrosis. This places individuals at risk of developing cirrhosis, hepatocellular carcinoma or hepatic decompensation and also contributes to the emergence of insulin resistance and dyslipidemias affecting many other organs. Excessive accumulation of TG in adipose tissue contributes to insulin resistance as well as to the release of cytokines attracting leucocytes leading to a pro-inflammatory state. Pathological accumulation of cholesteryl ester (CE) in macrophages in the arterial wall is the progenitor of atherosclerotic plaques and heart disease. Overconsumption of dietary fat, cholesterol and carbohydrates explains why these diseases are on the increase yet offers few clues for how to prevent or treat individuals. Dietary regimes have proven futile and barring surgery, no realistic alternatives are at hand as effective drugs are few and not without side effects. Overweight and obesity-related diseases are no longer restricted to the developed world and as such, constitute a global problem. Development of new drugs and treatment strategies are a priority yet requires as a first step, elucidation of the molecular pathophysiology underlying each associated disease state. The lipid droplet (LD), an up to now overlooked intracellular organelle, appears at the heart of each pathophysiology linking key regulatory and metabolic processes as well as constituting the site of storage of both TGs and CEs. As the molecular machinery and mechanisms of LDs of each cell type are being elucidated, regulatory proteins used to control various cellular processes are emerging. Of these and the subject of this review, small GTPases belonging to the Rab protein family appear as important molecular switches used in the regulation of the intracellular trafficking and storage of lipids.