Syntaxin 6: the promiscuous behaviour of a SNARE protein

Vesicle flow within the cell is responsible for the dynamic maintenance of and communication between intracellular compartments. In addition, vesicular transport is crucial for communication between the cell and its surrounding environment. The ability of a vesicle to recognise and fuse with an appropriate compartment or vesicle is determined by its protein and lipid composition as well as by proteins in the cytosol. SNARE proteins present on both vesicle as well as target organelle membranes provide one component necessary for the process of membrane fusion. While in mammalian cells the main focus of interest about SNARE function has centred on those involved in exocytosis, recent data on SNAREs involved in intracellular membrane-trafficking steps have provided a deeper insight into the properties of these proteins. We take, as an example, the promiscuous SNARE syntaxin 6, a SNARE involved in multiple membrane fusion events. The properties of syntaxin 6 reveal similarities but also differences in the behaviour of intracellular SNAREs and the highly specialised exocytotic SNARE molecules.     

Insulin Stimulates Syntaxin4 SNARE Complex Assembly via a Novel Regulatory Mechanism

Insulin stimulates glucose transport into fat and muscle cells by increasing the exocytic trafficking rate of the GLUT4 facilitative glucose transporter from intracellular stores to the plasma membrane. Delivery of GLUT4 to the plasma membrane is mediated by formation of functional SNARE complexes containing syntaxin4, SNAP23, and VAMP2. Here we have used an in situ proximity ligation assay to integrate these two observations by demonstrating for the first time that insulin stimulation causes an increase in syntaxin4-containing SNARE complex formation in adipocytes. Furthermore, we demonstrate that insulin brings about this increase in SNARE complex formation by mobilizing a pool of syntaxin4 held in an inactive state under basal conditions. Finally, we have identified phosphorylation of the regulatory protein Munc18c, a direct target of the insulin receptor, as a molecular switch to coordinate this process. Hence, this report provides molecular detail of how the cell alters membrane traffic in response to an external stimulus, in this case, insulin.     

Expanding the cellular molecular chaperone network through the ubiquitous cochaperones

Cellular environments are highly complex and contain a copious variety of proteins that must operate in unison to achieve homeostasis. To guide and preserve order, multifaceted molecular chaperone networks are present within each cell type. To handle the vast client diversity and regulatory demands, a wide assortment of chaperones are needed. In addition to the classic heat shock proteins, cochaperones with inherent chaperoning abilities (e.g., p23, Hsp40, Cdc37, etc.) are likely used to complete a system. In this review, we focus on the HSP90-associated cochaperones and provide evidence supporting a model in which select cochaperones are used to differentially modulate target proteins, contribute to combinatorial client regulation, and increase the overall reach of a cellular molecular chaperone network. This article is part of a Special Issue entitled: Heat Shock Protein 90 (HSP90).     

Unraveling secrets of telomeres: One molecule at a time

Telomeres play important roles in maintaining the stability of linear chromosomes. Telomere maintenance involves dynamic actions of multiple proteins interacting with long repetitive sequences and complex dynamic DNA structures, such as G-quadruplexes, T-loops and t-circles. Given the heterogeneity and complexity of telomeres, single-molecule approaches are essential to fully understand the structure–function relationships that govern telomere maintenance. In this review, we present a brief overview of the principles of single-molecule imaging and manipulation techniques. We then highlight results obtained from applying these single-molecule techniques for studying structure, dynamics and functions of G-quadruplexes, telomerase, and shelterin proteins.     

Unraveling the genomic mosaic of a ubiquitous genus of marine cyanobacteria

Background

The picocyanobacterial genus Synechococcus occurs over wide oceanic expanses, having colonized most available niches in the photic zone. Large scale distribution patterns of the different Synechococcus clades (based on 16S rRNA gene markers) suggest the occurrence of two major lifestyles ('opportunists'/'specialists'), corresponding to two distinct broad habitats ('coastal'/'open ocean'). Yet, the genetic basis of niche partitioning is still poorly understood in this ecologically important group.

Results

Here, we compare the genomes of 11 marine Synechococcus isolates, representing 10 distinct lineages. Phylogenies inferred from the core genome allowed us to refine the taxonomic relationships between clades by revealing a clear dichotomy within the main subcluster, reminiscent of the two aforementioned lifestyles. Genome size is strongly correlated with the cumulative lengths of hypervariable regions (or 'islands'). One of these, encompassing most genes encoding the light-harvesting phycobilisome rod complexes, is involved in adaptation to changes in light quality and has clearly been transferred between members of different Synechococcus lineages. Furthermore, we observed that two strains (RS9917 and WH5701) that have similar pigmentation and physiology have an unusually high number of genes in common, given their phylogenetic distance.

Conclusion

We propose that while members of a given marine Synechococcus lineage may have the same broad geographical distribution, local niche occupancy is facilitated by lateral gene transfers, a process in which genomic islands play a key role as a repository for transferred genes. Our work also highlights the need for developing picocyanobacterial systematics based on genome-derived parameters combined with ecological and physiological data.     

Unraveling helicase mechanisms one molecule at a time

Recent years have seen an increasing number of biological applications of single molecule techniques, evolving from a proof of principle type to the more sophisticated studies. Here we compare the capabilities and limitations of different single molecule techniques in studying the activities of helicases. Helicases share a common catalytic activity but present a high variability in kinetic and phenomenological behavior, making their studies ideal in exemplifying the use of the new single molecule techniques to answer biological questions. Unexpected phenomena have also been observed from individual molecules suggesting extended or alternative functionality of helicases in vivo.     

Tyrosine phosphorylation of a SNARE protein,Syntaxin 17: Implications for membrane trafficking in the early secretory pathway

The T-cell protein tyrosine phosphatase is expressed as two splice variants — TC45, a nuclear protein, and TC48, which is localized predominantly in the ER (endoplasmic reticulum). Yeast two-hybrid screening revealed direct interaction of TC48 with Syntaxin17, a SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein localized predominantly in the ER and to some extent in the ER-Golgi intermediate compartment. Syntaxin 17 did not interact with TC45. C-terminal 40 amino acids of TC48 were sufficient for interaction with syntaxin 17. Overexpressed syntaxin 17 was phosphorylated at tyrosine upon pervanadate treatment (a tyrosine phosphatase inhibitor/tyrosine kinase activator) of COS-1 cells. Mutational analysis identified Tyr156 in the cytoplasmic domain as the major site of phosphorylation. Endogenous syntaxin 17 was phosphorylated by pervanadate treatment in CHO and MIN6 cells but was not phosphorylated in a variety of other cell lines tested. c-Abl was identified as one of the kinases, which phosphorylates syntaxin 17 in MIN6 cells. Phosphorylation of endogenous and overexpressed syntaxin 17 was reduced in the presence of IGF receptor and EGF receptor kinase inhibitors. Serum depletion reduced pervanadate-induced phosphorylation of endogenous syntaxin 17. TC48 coexpression reduced phosphorylation of syntaxin 17 by pervanadate and purified TC48 directly dephosphorylated syntaxin 17. β-COP dispersal by overexpressed syntaxin 17 was reduced after pervanadate-induced phosphorylation. A phospho-mimicking mutant (Y156E) of syntaxin 17 showed reduced interaction with COPI vesicles. These results suggest that tyrosine phosphorylation of syntaxin 17 is likely to have a role in regulating syntaxin 17 dependent membrane trafficking in the early secretory pathway.     

GS32, a Novel Golgi SNARE of 32 kDa, Interacts Preferentially with Syntaxin 6

Syntaxin 1, synaptobrevins or vesicle-associated membrane proteins, and the synaptosome-associated protein of 25 kDa (SNAP-25) are key molecules involved in the docking and fusion of synaptic vesicles with the presynaptic membrane. We report here the molecular, cell biological, and biochemical characterization of a 32-kDa protein homologous to both SNAP-25 (20% amino acid sequence identity) and the recently identified SNAP-23 (19% amino acid sequence identity). Northern blot analysis shows that the mRNA for this protein is widely expressed. Polyclonal antibodies against this protein detect a 32-kDa protein present in both cytosol and membrane fractions. The membrane-bound form of this protein is revealed to be primarily localized to the Golgi apparatus by indirect immunofluorescence microscopy, a finding that is further established by electron microscopy immunogold labeling showing that this protein is present in tubular-vesicular structures of the Golgi apparatus. Biochemical characterizations establish that this protein behaves like a SNAP receptor and is thus named Golgi SNARE of 32 kDa (GS32). GS32 in the Golgi extract is preferentially retained by the immobilized GST–syntaxin 6 fusion protein. The coimmunoprecipitation of syntaxin 6 but not syntaxin 5 or GS28 from the Golgi extract by antibodies against GS32 further sustains the preferential interaction of GS32 with Golgi syntaxin 6.     

Munc18-1 and Syntaxin1: Unraveling the Interactions Between the Dynamic Duo

All neurotransmitter and hormone regulated secretory events involve the action of three soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, syntaxin, SNAP-25, and synaptobrevin. The SNARE proteins interact to form a four alpha-helical complex, involving syntaxin and SNAP-25 on the plasma membrane and synaptobrevin on the vesicular membrane, bringing the opposing membranes together, promoting bilayer merger and membrane fusion. The process of regulated secretion is an adaptation of the membrane fusion events which occur at multiple steps throughout the intracellular trafficking pathway, in each case catalyzed by SNARE protein isoforms. At all of these locations, the SNAREs are joined by a member of the Sec1p/Munc18 (SM) protein family which selectively bind to syntaxin isoforms. From their initial identification, the SM proteins were known to be essential for membrane fusion, however, over the intervening decades, deciphering the precise mechanism of action of the SM proteins has proved problematic. Recent studies, investigating the interactions of munc18-1 and syntaxin1, provide an explanation for previous, apparently conflicting, observations yielding a new understanding of their cellular functions.     

Arrestins: ubiquitous regulators of cellular signaling pathways

In vertebrates, the arrestins are a family of four proteins that regulate the signaling and trafficking of hundreds of different G-protein-coupled receptors (GPCRs). Arrestin homologs are also found in insects, protochordates and nematodes. Fungi and protists have related proteins but do not have true arrestins. Structural information is available only for free (unbound) vertebrate arrestins, and shows that the conserved overall fold is elongated and composed of two domains, with the core of each domain consisting of a seven-stranded β-sandwich. Two main intramolecular interactions keep the two domains in the correct relative orientation, but both of these interactions are destabilized in the process of receptor binding, suggesting that the conformation of bound arrestin is quite different. As well as binding to hundreds of GPCR subtypes, arrestins interact with other classes of membrane receptors and more than 20 surprisingly diverse types of soluble signaling protein. Arrestins thus serve as ubiquitous signaling regulators in the cytoplasm and nucleus.     

Interaction of the conserved oligomeric Golgi complex with t-SNARE Syntaxin5a/Sed5 enhances intra-Golgi SNARE complex stability

Tethering factors mediate initial interaction of transport vesicles with target membranes. Soluble N-ethylmaleimide–sensitive fusion protein attachment protein receptors (SNAREs) enable consequent docking and membrane fusion. We demonstrate that the vesicle tether conserved oligomeric Golgi (COG) complex colocalizes and coimmunoprecipitates with intra-Golgi SNARE molecules. In yeast cells, the COG complex preferentially interacts with the SNARE complexes containing yeast Golgi target (t)-SNARE Sed5p. In mammalian cells, hCog4p and hCog6p interact with Syntaxin5a, the mammalian homologue of Sed5p. Moreover, fluorescence resonance energy transfer reveals an in vivo interaction between Syntaxin5a and the COG complex. Knockdown of the mammalian COG complex decreases Golgi SNARE mobility, produces an accumulation of free Syntaxin5, and decreases the steady-state levels of the intra-Golgi SNARE complex. Finally, overexpression of the hCog4p N-terminal Syntaxin5a-binding domain destabilizes intra-Golgi SNARE complexes, disrupting the Golgi. These data suggest that the COG complex orchestrates vesicular trafficking similarly in yeast and mammalian cells by binding to the t-SNARE Syntaxin5a/Sed5p and enhancing the stability of intra-Golgi SNARE complexes.     

Syntaxin 16 is enriched in neuronal dendrites and may have a role in neurite outgrowth

Polarized membrane traffic to different domains of the neuron is well documented, and is required for both establishment and maintenance of neuronal polarity. Some soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins, particularly syntaxin 12/13 and TI-VAMP/VAMP7, have known roles in the neuron. We report here that the brain-enriched SNARE syntaxin 16 (Syn 16) is specifically enriched in neuronal dendrites and found at Golgi outposts, thus confirming that Golgi outposts are endowed with a trans-Golgi network (TGN) component. Over-expression of wild type syntaxin 16 moderately stimulates, whereas that of an N-terminal deletion mutant (Syn 16-DeltaNt) inhibits, neurite outgrowth in both mouse Neuro-2a cells and primary cortical neurons. Consistent with an inhibited neurite growth, cells over-expressing Syn 16-DeltaNt have diminished betaIII-tubulin and F-actin labeling. RNA interference-mediated silencing of syntaxin 16 in primary cortical neurons significantly retards neurite outgrowth. Syntaxin 16 may thus play a role in neurite outgrowth and perhaps other specific dendritic anterograde/retrograde traffic.     

Fibromodulin: A regulatory molecule maintaining cellular architecture for normal cellular function

Fibromodulin (FMOD) is a small leucine-rich proteoglycan that plays roles in a series of biological and pathophysiological processes. The interaction between FMOD and lysyl oxidase (LOX; collagen cross-linking enzyme) helps regulate extracellular matrix composition, and thereby, provides a permissive environment for regulating cellular turnover. FMOD has been mostly studied in the context of matrix component assembly, but during recent years its association with muscle development, cell reprogramming, and the angiogenic program have demonstrated its activities well beyond extracellular matrix maintenance. In fact, the involvement of FMOD in these cellular processes places it the centrum of cellular behaviour and ultimately of tissue properties. Thus, a clear view of the impact FMOD has on tissue integrity would aid its exploitation for tissue modelling and in the treatment of different disorders.     

Cyclic diguanylate is a ubiquitous signaling molecule in bacteria: insights into biochemistry of the GGDEF protein domain

Proteins containing GGDEF domains are encoded in the majority of sequenced bacterial genomes. In several species, these proteins have been implicated in biosynthesis of exopolysaccharides, formation of biofilms, establishment of a sessile lifestyle, surface motility, and regulation of gene expression. However, biochemical activities of only a few GGDEF domain proteins have been tested. These proteins were shown to be involved in either synthesis or hydrolysis of cyclic-bis(3'-->5') dimeric GMP (c-di-GMP) or in hydrolysis of cyclic AMP. To investigate specificity of the GGDEF domains in Bacteria, six GGDEF domain-encoding genes from randomly chosen representatives of diverse branches of the bacterial phylogenetic tree, i.e., Thermotoga, Deinococcus-Thermus, Cyanobacteria, spirochetes, and alpha and gamma divisions of the Proteobacteria, were cloned and overexpressed. All recombinant proteins were purified and found to possess diguanylate cyclase (DGC) activity involved in c-di-GMP synthesis. The individual GGDEF domains from two proteins were overexpressed, purified, and shown to possess a low level of DGC activity. The oligomeric states of full-length proteins and individual GGDEF domains were similar. This suggests that GGDEF domains are sufficient to encode DGC activity; however, enzymatic activity is highly regulated by the adjacent sensory protein domains. It is shown that DGC activity of the GGDEF domain protein Rrp1 from Borrelia burgdorferi is strictly dependent on phosphorylation status of its input receiver domain. This study establishes that majority of GGDEF domain proteins are c-di-GMP specific, that c-di-GMP synthesis is a wide-spread phenomenon in Bacteria, and that it is highly regulated.     

BRCA1-hapoinsufficiency: Unraveling the molecular and cellular basis for tissue-specific cancer

Over the past 20 years tremendous progress has been made in understanding the function of BRCA1 gene products. Yet one question still remains: why is mutation of BRCA1 typically associated with preferential development of breast and ovarian cancers and not tumors in other tissues? Here we discuss recent evidence documenting the effect of BRCA1-haploinsufficiency in different cells and tissues and synthesize a model for how mutations in a single BRCA1 allele in human cells might preferentially confer increased cancer risk in breast epithelial cells.