Synaptic proteins and SNARE complexes are localized in lipid rafts from rat brain synaptosomes

The biochemical characterization of the SNARE proteins present in lipid microdomains, also known as "lipid rafts," has been addressed in earlier studies, with conflicting data from different laboratories. In this study, we use rat brain synaptosomes as a model with which to examine the presence of proteins involved in exocytosis in detergent-resistant membranes (DRM), also known as 'lipid rafts.' By means of buoyancy analysis in sucrose gradients of Triton X-100-solubilized synaptosomes, we identified a pool of SNARE proteins (SNAP 25, syntaxin 1, and synaptobrevin2/VAMP2) significantly associated with DRM. Furthermore, Munc18, synaptophysin, and high amounts of the isoforms I and II of synaptotagmin were also found in DRM. In addition, SDS-resistant and temperature-dependent SNARE complexes were also detected in DRM. Treatment of synaptosomes with methyl-beta-cyclodextrin resulted in persistence of the proteins present in the DRM isolated using Triton X-100, whilst strongly impairing calcium-dependent glutamate release. The results from the present work show that lipid microdomains are sites where SNARE proteins and complexes are actually present, as well as important elements in the control of regulated exocytosis.     

Triglyceride containing lipid droplets and lipid droplet-associated proteins

PURPOSE OF REVIEW: Cytosolic lipid droplets are now recognized as dynamic organelles. This review summarizes our current understanding of the mechanisms involved in the formation of lipid droplets, the importance of lipid droplet-associated proteins and the link between lipid droplet accumulation and development of insulin resistance. RECENT FINDINGS: Lipid droplets are formed as primordial droplets and they increase in size by fusion. This fusion process requires the alpha-soluble N-ethylmaleimide-sensitive factor adaptor protein receptor SNAP23, which is also involved in the insulin-dependent translocation of a glucose transporter to the plasma membrane. Recent data suggest that SNAP23 is the link between increased lipid droplet accumulation and development of insulin resistance. Lipid droplets also form tight interactions with other organelles. Furthermore, additional lipid droplet-associated proteins have been identified and shown to play a role in droplet assembly and turnover, and in sorting and trafficking events. SUMMARY: Recent studies have identified a number of key proteins that are involved in the formation and turnover of lipid droplets, and SNAP23 has been identified as a link between accumulation of lipid droplets and development of insulin resistance. Further understanding of lipid droplet biology could indicate potential therapeutic targets to prevent accumulation of lipid droplets and associated complications.     

Demonstrated and inferred metabolism associated with cytosolic lipid droplets

Cytosolic lipid droplets were considered until recently to be rather inert particles of stored neutral lipid. Largely through proteomics is it now known that droplets are dynamic organelles and that they participate in several important metabolic reactions as well as trafficking and interorganellar communication. In this review, the role of droplets in metabolism in the yeast Saccharomyces cerevisiae, the fly Drosophila melanogaster, and several mammalian sources are discussed, particularly focusing on those reactions shared by these organisms. From proteomics and older work, it is clear that droplets are important for fatty acid and sterol biosynthesis, fatty acid activation, and lipolysis. However, many droplet-associated enzymes are predicted to span a membrane two or more times, which suggests either that droplet structure is more complex than the current model posits, or that there are tightly bound membranes, particularly derived from the endoplasmic reticulum, which account for the association of several of these proteins.Cytosolic lipid droplets, originally thought to be simply coalesced neutral lipids waiting for lipolysis at metabolic demand, are now known to be considerably more complicated both structurally and functionally. There is general agreement that droplets are comprised of a core of neutral lipids, principally triglycerides and steryl esters, surrounded by a leaflet of phospholipids into which are embedded a specific subset of cellular proteins, the most abundant of which are members of the PAT family (see below) in animal cells (1). However, this model is probably too simple; there is evidence from physical probes of droplets isolated from yeast mutants unable to synthesize triglycerides or steryl esters that these two molecular families are partially segregated within the core, with thin shells of steryl esters forming concentric hollow spheres around an inner core composed principally of triglycerides (2).The next layer of complexity is the functional inhomogeneity of droplets. Subsets of droplets within the same cells exist with different populations of PAT proteins, differentiating among different sizes, ages, and levels of metabolic activity (3, 4). Perhaps most surprisingly, droplets may be comprised, at least in some cases, not of the layered core-phospholipid shell architecture at all but a knot of tightly woven endoplasmic reticulum (ER) surrounded by secreted neutral lipid, itself encased with a single leaflet. Such a model is based on electron microscopic thin sections (5), freeze fracture-immunogold evidence (6), immunohistochemical studies of ER luminal proteins within the droplet (7), and the identification of these proteins, notably ER chaperones, in several proteomic studies. Although certainly, such a complex structure must obey physical laws governing aqueous interactions with hydrophobic lipids and artifacts in processing for electron microscopy do occur, it may be best at present to keep an open mind and consider that droplets may not have the same structure among tissues and that they may take multiple physical forms in rapid order as they dynamically perform their functions.What are these functions? The most obvious one is lipid metabolism, namely the biogenesis and breakdown of the neutral lipids contained within the droplet. Although this conclusion predates proteomic studies (8), these recent studies have revealed the breadth and conservation of metabolic reactions that occur at or near the droplet surface, the subject of this review. Moreover, proteomics has demonstrated the surprising fact that droplets are likely to be very active in organellar communication because they are replete in rab proteins and other trafficking molecules. Our knowledge from proteomic studies of droplet trafficking and communication is discussed separately in this thematic review series.A major caveat must be kept in mind when evaluating droplet proteomics data: besides droplet trafficking through transient interactions with vesicles or target organelles such as early endosomes (9), droplets make extensive, tight, and long-lasting synapses with the endoplasmic reticulum, mitochondria, and peroxisomes (10, 11). The fact that ER, mitochondrial, peroxisomal, and a few plasma membrane proteins are found with such high frequency in the droplet proteome probably reflects these tight interorganellar interactions, perhaps similar to the mitochondrially associated membranes (MAMs) that link mitochondria with ER (12). The molecular basis for droplet-mediated synapses are not yet known. Besides the frequent occurrence of specific nondroplet organelle proteins in the droplet proteome, adventitious contamination of droplets is unlikely in view of the unique density of droplets that allow their flotation to the top of aqueous buffers and density gradients after centrifugation while all other cell components sink (which also permits several washes with high recovery), and the nonrandom coisolation of subsets of proteins from other organelles, such as the β-oxidation peroxisomal enzymes (10), which suggests specialized regions for metabolically-productive droplet interactions at the synapses.Droplet-ER interactions are a special case; it is the rule rather than the exception that enzymes of lipid metabolism that are found in the droplet proteome are also found to varying extents in the ER. This has been well documented in yeast through genome-wide green fluorescent protein (GFP)-tagging (13, 14). Erg6p, an enzyme in the latter part of the ergosterol biosynthetic pathway, is the only droplet protein in the pathway with a near-exclusive droplet localization in yeast; Erg1p, Erg7p, and Erg 27p are dually localized, and the pattern changes depending on metabolic state. Whether this general rule is specific for yeast, in which droplets remain on the ER surface (15), is not yet clear. However, several examples already exist in mammalian cells: cytochrome b5 reductase (DT diaphorase) and various sterol dehydrogenases (see Table 1), were classically considered ER proteins. Many enzymes of sterol metabolism that appear in droplet proteomes have multiple membrane spans and it is difficult to imagine them arranged in the single leaflet surrounding a hydrophobic core of neutral lipids. A solution to this problem, besides that of invoking internal ER cisternae within droplets, is to consider these enzymes in a specialized ER compartment that is very close to, and tightly bound with, the droplet (a “droplet synapse”) that separates from the bulk ER during fractionation, copurifying with droplets. If this structure resembles that of MAMs in contact with mitochondria, it would explain the frequent coisolation of ER luminal chaperones with droplets because chaperones such as luminal HSP70 are directly involved in MAM structure (12).

TABLE 1.

Metabolic functions of droplets as revealed by proteomics

Proteomics of photoreceptor outer segments identifies a subset of SNARE and Rab proteins implicated in membrane vesicle trafficking and fusion

The outer segment is a specialized compartment of vertebrate rod and cone photoreceptor cells where phototransduction takes place. In rod cells it consists of an organized stack of disks enclosed by a separate plasma membrane. Although most proteins involved in phototransduction have been identified and characterized, little is known about the proteins that are responsible for outer segment structure and renewal. In this study we used a tandem mass spectrometry-based proteomics approach to identify proteins in rod outer segment preparations as an initial step in defining their roles in photoreceptor structure, function, renewal, and degeneration. Five hundred and sixteen proteins were identified including 41 proteins that function in rod and cone phototransduction and the visual cycle and most proteins previously shown to be involved in outer segment structure and metabolic pathways. In addition, numerous proteins were detected that have not been previously reported to be present in outer segments including a subset of Rab and SNARE proteins implicated in vesicle trafficking and membrane fusion. Western blotting and immunofluorescence microscopy confirmed the presence of Rab 11b, Rab 18, Rab 1b, and Rab GDP dissociation inhibitor in outer segments. The SNARE proteins, VAMP2/3, syntaxin 3, N-ethylmaleimide-sensitive factor, and Munc 18 detected in outer segment preparations by mass spectrometry and Western blotting were also observed in outer segments by immunofluorescence microscopy. Syntaxin 3 and N-ethylmaleimide- sensitive factor had a restricted localization at the base of the outer segments, whereas VAMP2/3 and Munc 18 were distributed throughout the outer segments. These results suggest that Rab and SNARE proteins play a role in vesicle trafficking and membrane fusion as part of the outer segment renewal process. The data set generated in this study is a valuable resource for further analysis of photoreceptor outer segment structure and function.     

A novel SNARE complex implicated in vesicle fusion with the endoplasmic reticulum.

Intracellular vesicular traffic is controlled in part by v- and t-SNAREs, integral membrane proteins which allow specific interaction and fusion between vesicles (v-SNAREs) and their target membranes (t-SNAREs). In yeast, retrograde transport from the Golgi complex to the ER is mediated by the ER t-SNARE Ufe1p, and also requires two other ER proteins, Sec20p and Tip20p, which bind each other. Although Sec20p is not a typical SNARE, we show that both it and Tip20p can be co-precipitated with Ufe1p, and that a growth-inhibiting mutation in Ufe1p can be compensated by a mutation in Sec20p. Furthermore, Sec22p, a v-SNARE implicated in forward transport from ER to Golgi, co-precipitates with Ufe1p and Sec20p, and SEC22 acts as an allele-specific multicopy suppressor of a temperature-sensitive ufe1 mutation. These results define a new functional SNARE complex, with features distinct from the plasma membrane and cis-Golgi complexes previously identified. They also show that a single v-SNARE can be involved in both anterograde and retrograde transport, which suggests that the mere presence of a particular v-SNARE may not be sufficient to determine the preferred target for a transport vesicle.     

Sec17p and HOPS, in distinct SNARE complexes, mediate SNARE complex disruption or assembly for fusion

SNARE functions during membrane docking and fusion are regulated by Sec1/Munc18 (SM) chaperones and Rab/Ypt GTPase effectors. These functions for yeast vacuole fusion are combined in the six-subunit HOPS complex. HOPS facilitates Ypt7p nucleotide exchange, is a Ypt7p effector, and contains an SM protein. We have dissected the associations and requirements for HOPS, Ypt7p, and Sec17/18p during SNARE complex assembly. Vacuole SNARE complexes bind either Sec17p or the HOPS complex, but not both. Sec17p and its co-chaperone Sec18p disassemble SNARE complexes. Ypt7p regulates the reassembly of unpaired SNAREs with each other and with HOPS, forming HOPS.SNARE complexes prior to fusion. After HOPS.SNARE assembly, lipid rearrangements are still required for vacuole content mixing. Thus, Sec17p and HOPS have mutually exclusive interactions with vacuole SNAREs to mediate disruption of SNARE complexes or their assembly for docking and fusion. Sec17p may displace HOPS from SNAREs to permit subsequent rounds of fusion.     

Cell membrane lipid rafts mediate caveolar endocytosis of HIV-1 Tat fusion proteins

The transactivator protein of human immunodeficiency virus type 1 Tat has the unique property of mediating the delivery of large protein cargoes into the cells when present in the extracellular milieu. Here we show that Tat fusion proteins are internalized by the cells through a temperature-dependent endocytic pathway that originates from cell membrane lipid rafts and follows caveolar endocytosis. These conclusions are supported by the study of the slow kinetics of the internalization of Tat endosomes, by their resistance to nonionic detergents, the colocalization of internalized Tat with markers of caveolar endocytosis, and the impairment of the internalization process by drugs that disrupt lipid rafts or disturb caveolar trafficking. These results are of interest for all those who exploit Tat as a vehicle for transcellular protein delivery.     

The vesicle- and target-SNARE proteins that mediate Glut4 vesicle fusion are localized in detergent-insoluble lipid rafts present on distinct intracellular membranes

Insulin stimulates the fusion of intracellular vesicles containing the glucose transporter Glut4 with the plasma membrane in adipocytes and muscle cells. Glut4 vesicle fusion is thought to be catalyzed by the interaction of the vesicle soluble N-ethyl-maleimide-sensitive fusion protein attachment protein receptor VAMP2 with the target soluble N-ethyl-maleimide-sensitive fusion protein attachment protein receptors SNAP-23 and syntaxin 4. Here, we use combined membrane fractionation, detergent solubility, and sucrose gradient flotation to demonstrate that the large majority (>70%) of SNAP-23 and a significant proportion of syntaxin 4 ( approximately 35%) are associated with plasma membrane lipid rafts in 3T3-L1 adipocytes. Furthermore, VAMP2 is shown to be concentrated in lipid rafts isolated from intracellular membranes. Insulin stimulation had no effect on the plasma membrane raft association of SNAP-23 or syntaxin 4 but promoted VAMP2 insertion into plasma membrane rafts. Immunofluorescence analysis revealed that SNAP-23 was clustered at the plasma membrane and almost completely segregated from the transferrin receptor. SNAP-23 distribution seemed to be distinct from caveolin-1, and clusters of SNAP-23 were dispersed after cholesterol extraction with methyl-beta-cyclodextrin, suggesting that the majority of SNAP-23 is associated with non-caveolar, cholesterol-rich lipid rafts. The results described implicate lipid rafts as important platforms for Glut4 vesicle fusion and suggest the hypothesis that such rafts may represent a spatial integration point of insulin signaling and membrane traffic.     

SNARE proteins are not excessive for the formation of post-Golgi SNARE complexes in HeLa cells

Regulators of G protein signaling (RGS proteins) serve as GTPase activating proteins for the signal transducing Gα subunits. RGS19, also known as Gα-interacting protein (GAIP), has been shown to subserve other functions such as the regulation of macroautophagy and growth factor signaling. We have recently demonstrated that the expression of RGS19 in human embryonic kidney (HEK) 293 cells resulted in the disruption of serum-induced mitogenic response along the classical Ras/Raf/MEK/ERK pathway. Here, we further examined the effect of RGS19 expression on the stress-activated protein kinases (SAPKs). Both c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) became non-responsive to serum in 293/RGS19 cells, yet the two SAPKs responded to UV irradiation or osmotic stress induced by sorbitol. Kinases upstream of JNK and p38 MAPK, including MKK3/6, MKK4, and MLK3, also failed to respond to serum stimulation in 293/RGS19 cells. Serum-induced activation of the small GTPases Rac1 and Cdc42 was similarly suppressed in these cells. Our results indicate that elevated expression of RGS19 can severely disrupt the regulation of MAPKs by small GTPases.     

Alternate interfaces may mediate homomeric and heteromeric assembly in the transmembrane domains of SNARE proteins

The fusion of a vesicle to a target membrane is mediated by temporally and spatially regulated interactions within a set of evolutionarily conserved proteins. Integral to proper fusion is the interaction between proteins originating on both vesicle and target membranes to form a protein bridge between the two membranes, known as the SNARE complex. This protein complex includes the single-pass transmembrane helix proteins: syntaxin and synaptobrevin. Experimental data and amino acid sequence analysis suggest that an interface of interaction is conserved between the transmembrane regions of the two proteins. However, conflicting reports have been presented on the role of the synaptobrevin transmembrane domain in mediating important protein-protein interactions. To address this question, a thermodynamic study was carried out to determine quantitatively the self-association propensities of the transmembrane domains of synaptobrevin and syntaxin. Our results show that the transmembrane domain of synaptobrevin has only a modest ability to self-associate, whereas the transmembrane domain of syntaxin is able to form stable homodimers. Nevertheless, by a single amino acid substitution, synaptobrevin can be driven to dimerize with the same affinity as syntaxin. Furthermore, crosslinking studies show that dimerization of synaptobrevin is promoted by oxidizing agents. Despite the presence of a conserved cysteine residue in the same location as in synaptobrevin, syntaxin dimerization is not promoted by oxidization. This analysis suggests that subtle yet distinct differences are present between the two transmembrane dimer interfaces. A syntaxin/synaptobrevin heterodimer is able to form under oxidizing conditions, and we propose that the interface of interaction for the heterodimer may resemble the homodimer interface formed by the synaptobrevin transmembrane domain. Computational analysis of the transmembrane sequences of syntaxin and synaptobrevin reveal structural models that correlate with the experimental data. These data may provide insight into the role of transmembrane segments in the mechanism of vesicle fusion.     

Normal insulin sensitivity and IMCL content in overweight humans are associated with higher fasting lipid oxidation

Intramyocellular lipid (IMCL) storage is considered a local marker of whole body insulin resistance; because increments of body weight are supposed to impair insulin sensitivity, this study was designed to assess IMCL content, lipid oxidation, and insulin action in individuals with a moderate increment of body fat mass and no family history of diabetes. We studied 14 young, nonobese women with body fat <30% (n = 7) or >30% (n = 7) and 14 young, nonobese men with body fat <25% (n = 7) or >25% (n = 7) by means of the euglycemic-insulin clamp to assess whole body glucose metabolism, with indirect calorimetry to assess lipid oxidation, by localized (1)H NMR spectroscopy of the calf muscles to assess IMCL content, and with dual-energy X-ray absorptiometry to assess body composition. Subjects with higher body fat had normal insulin-stimulated glucose disposal (P = 0.80), IMCL content in both soleus (P = 0.22) and tibialis anterior (P = 0.75) muscles, and plasma free fatty acid levels (P = 0.075) compared with leaner subjects in association with increased lipid oxidation (P < 0.05), resting energy expenditure (P = 0.046), resting oxygen consumption (P = 0.049), and plasma leptin levels (P < 0.01) in the postabsorptive condition. In conclusion, in overweight subjects, preservation of insulin sensitivity was combined with increased lipid oxidation and maintenance of normal IMCL content, suggesting that abnormalities of these factors may mutually determine the development of insulin resistance associated with weight gain.     

Ternary SNARE complexes are enriched in lipid rafts during mast cell exocytosis

Lipid rafts are membrane microdomains rich in cholesterol and glycosphingolipids that have been implicated in the regulation of intracellular protein trafficking. During exocytosis, a class of proteins termed SNAREs mediate secretory granule-plasma membrane fusion. To investigate the role of lipid rafts in secretory granule exocytosis, we examined the raft association of SNARE proteins and SNARE complexes in rat basophilic leukemia (RBL) mast cells. The SNARE protein SNAP-23 co-localized with a lipid raft marker and was present in detergent-insoluble lipid raft microdomains in RBL cells. By contrast, only small amounts (<20%) of the plasma membrane SNARE syntaxin 4 or the granule-associated SNARE vesicle-associated membrane protein (VAMP)-2 were present in these microdomains. Despite this, essentially all syntaxin 4 and most of VAMP-2 in these rafts were present in SNARE complexes containing SNAP-23, while essentially none of these complexes were present in nonraft membranes. Whereas SNAP-23 is membrane anchored by palmitoylation, the association of the transmembrane protein syntaxin 4 with lipid rafts was because of its binding to SNAP-23. After stimulating mast cells exocytosis, the amount of syntaxin 4 and VAMP-2 present in rafts increased twofold, and these proteins were now present in raft-associated phospho-SNAP-23/syntaxin 4/VAMP-2 complexes, revealing differential association of SNARE fusion complexes during the process of regulated exocytosis.     

Monocyte lipid rafts contain proteins implicated in vesicular trafficking and phagosome formation

Lipid rafts are membrane microdomains of unique lipid composition that segregate proteins with poorly understood consequences for membrane organization. Identification of raft associated proteins could therefore provide novel insight into raft-dependent functions. Monocytes process antigens for presentation to T cells by ingesting pathogens into calcium-dependent plasma membrane invaginations called "phagosomes" which develop by sequential fusion with the endoplasmic reticulum, early and late endosomes. We investigated the protein composition of Triton X-100 insoluble low density membranes of the monocyte cell-line THP-1 by matrix-assisted laser desorption/ionization-time of flight and tandem mass spectrometry. The ganglioside GM1 colocalized on the plasma membrane with the raft markers flotillin 1 and 2, which were enriched in low buoyant density fractions containing 52 identifiable proteins, 28 of which have not been reported in rafts, and nine of which are associated with the endoplasmic reticulum (ER). Remarkably, 27 of the 52 proteins are components of phagosomes, including the ER protein calnexin which we demonstrate is phosphorylated on serine 562, a switch controlling calcium homeostasis. The presence of the early and late endosome trafficking proteins Rab-1, and Rab-7 together with the late endosome protein LIMPII, indicate lipid rafts are present throughout endosome maturation. Identification of vacuolar ATP synthase, and synaptosomal-associated protein-23, proteins implicated in membrane fusion, together with the cytoskeletal proteins actin, alpha-actinin, and vimentin, and Rac 1, 2, and 3, regulators of cytoskeletal assembly, indicate monocyte lipid rafts contain the machinery to direct vesicular fusion and actin based vesicular migration throughout phagosome development.     

Lysosomal fusion and SNARE function are impaired by cholesterol accumulation in lysosomal storage disorders

The function of lysosomes relies on the ability of the lysosomal membrane to fuse with several target membranes in the cell. It is known that in lysosomal storage disorders (LSDs), lysosomal accumulation of several types of substrates is associated with lysosomal dysfunction and impairment of endocytic membrane traffic. By analysing cells from two severe neurodegenerative LSDs, we observed that cholesterol abnormally accumulates in the endolysosomal membrane of LSD cells, thereby reducing the ability of lysosomes to efficiently fuse with endocytic and autophagic vesicles. Furthermore, we discovered that soluble N‐ethylmaleimide‐sensitive factor attachment protein (SNAP) receptors (SNAREs), which are key components of the cellular membrane fusion machinery are aberrantly sequestered in cholesterol‐enriched regions of LSD endolysosomal membranes. This abnormal spatial organization locks SNAREs in complexes and impairs their sorting and recycling. Importantly, reducing membrane cholesterol levels in LSD cells restores normal SNARE function and efficient lysosomal fusion. Our results support a model by which cholesterol abnormalities determine lysosomal dysfunction and endocytic traffic jam in LSDs by impairing the membrane fusion machinery, thus suggesting new therapeutic targets for the treatment of these disorders.     

DGAT enzymes are required for triacylglycerol synthesis and lipid droplets in adipocytes

The total contribution of the acyl CoA:diacylglycerol acyltransferase (DGAT) enzymes, DGAT1 and DGAT2, to mammalian triacylglycerol (TG) synthesis has not been determined. Similarly, whether DGAT enzymes are required for lipid droplet (LD) formation is unknown. In this study, we examined the requirement for DGAT enzymes in TG synthesis and LDs in differentiated adipocytes with genetic deletions of DGAT1 and DGAT2. Adipocytes with a single deletion of either enzyme were capable of TG synthesis and LD formation. In contrast, adipocytes with deletions of both DGATs were severely lacking in TG and did not have LDs, indicating that DGAT1 and DGAT2 account for nearly all TG synthesis in adipocytes and appear to be required for LD formation during adipogenesis. DGAT enzymes were not absolutely required for LD formation in mammalian cells, however; macrophages deficient in both DGAT enzymes were able to form LDs when incubated with cholesterol-rich lipoproteins. Although adipocytes lacking both DGATs had no TG or LDs, they were fully differentiated by multiple criteria. Our findings show that DGAT1 and DGAT2 account for the vast majority of TG synthesis in mice, and DGAT function is required for LDs in adipocytes, but not in all cell types.     

SNARE proteins VAMP721 and VAMP722 mediate the post-Golgi trafficking required for auxin-mediated development in Arabidopsis

The plant hormone auxin controls many aspects of plant development. Membrane trafficking processes, such as secretion, endocytosis and recycling, regulate the polar localization of auxin transporters in order to establish an auxin concentration gradient. Here, we investigate the function of the Arabidopsis thaliana R-SNAREs VESICLE-ASSOCIATED MEMBRANE PROTEIN 721 (VAMP721) and VAMP722 in the post-Golgi trafficking required for proper auxin distribution and seedling growth. We show that multiple growth phenotypes, such as cotyledon development, vein patterning and lateral root growth, were defective in the double homozygous vamp721 vamp722 mutant. Abnormal auxin distribution and root patterning were also observed in the mutant seedlings. Fluorescence imaging revealed that three auxin transporters, PIN-FORMED 1 (PIN1), PIN2 and AUXIN RESISTANT 1 (AUX1), aberrantly accumulate within the cytoplasm of the double mutant, impairing the polar localization at the plasma membrane (PM). Analysis of intracellular trafficking demonstrated the involvement of VAMP721 and VAMP722 in the endocytosis of FM4-64 and the secretion and recycling of the PIN2 transporter protein to the PM, but not its trafficking to the vacuole. Furthermore, vamp721 vamp722 mutant roots display enlarged trans-Golgi network (TGN) structures, as indicated by the subcellular localization of a variety of marker proteins and the ultrastructure observed using transmission electron microscopy. Thus, our results suggest that the R-SNAREs VAMP721 and VAMP722 mediate the post-Golgi trafficking of auxin transporters to the PM from the TGN subdomains, substantially contributing to plant growth.     

Reconstitution of SNARE proteins into solid-supported lipid bilayer stacks and X-ray structure analysis

SNAREs are known as an important family of proteins mediating vesicle fusion. For various biophysical studies, they have been reconstituted into supported single bilayers via proteoliposome adsorption and rupture. In this study we extended this method to the reconstitution of SNAREs into supported multilamellar lipid membranes, i.e. oriented multibilayer stacks, as an ideal model system for X-ray structure analysis (X-ray reflectivity and diffraction). The reconstitution was implemented through a pathway of proteomicelle, proteoliposome and multibilayer. To monitor the structural evolution in each step, we used small-angle X-ray scattering for the proteomicelles and proteoliposomes, followed by X-ray reflectivity and grazing-incidence small-angle scattering for the multibilayers. Results show that SNAREs can be successfully reconstituted into supported multibilayers, with high enough orientational alignment for the application of surface sensitive X-ray characterizations. Based on this protocol, we then investigated the effect of SNAREs on the structure and phase diagram of the lipid membranes. Beyond this application, this reconstitution protocol could also be useful for X-ray analysis of many further membrane proteins.     

Altered insulin sensitivity, insulin secretion and lipid profile in non-diabetic prostate carcinoma

Insulin can influence cancer risk through its effect on cell proliferation, differentiation and apoptosis. Although hyperinsulinemia is considered as a risk factor in the pathogenesis of various cancers, the data related to insulin sensitivity, insulin secretion and lipid profile is lacking in non-diabetic prostate carcinoma cases. The present study was undertaken to evaluate lipid profile parameters and insulin sensitivity and secretion using surrogate markers derived from the measurements of fasting glucose and fasting insulin. The study group comprises 27 prostate carcinoma cases and 27 controls having similar age. Fasting serum insulin, glucose and lipid profile parameters were estimated in both the groups. Insulin sensitivity was assessed by Homeostasis model assessment of insulin sensitivity and Quantitative insulin sensitivity check index. Insulin secretion was assessed by insulinogenic index. Fasting serum insulin, insulinogenic index and LDL-cholesterol were significantly increased (p < 0.05) and HOMA-IS, QUICKI and HDL-cholesterol was significantly decreased (p < 0.05) in carcinoma cases compared to controls. PSA level was significantly associated with fasting insulin (R2 = 0.150, beta = 0.387, p = 0.046) and QUICKI (R2 = 0.173, beta = -0.416, p = 0.031). Fasting insulin was significantly correlated with triglyceride (r = 0.404, p = 0.037) and HDL-cholesterol (r = -0.474, p = 0.013). The present study concludes that hyperinsulinemia associated with reduced insulin sensitivity may play a role in the pathogenesis of prostate carcinoma.