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1.  To protect or reject 
eLife  2014;3:e03374.
A protein known for its role in dismantling faulty SNARE complexes can also help to maintain complexes that have formed properly during membrane fusion.
doi:10.7554/eLife.03374
PMCID: PMC4060003  PMID: 24940001
membrane; SNARE; Docking; HOPS; lysosome; Golgi; S. cerevisiae
2.  Exorcising the Exocyst Complex 
Traffic (Copenhagen, Denmark)  2012;13(7):898-907.
The exocyst complex is an evolutionarily conserved multisubunit protein complex implicated in tethering secretory vesicles to the plasma membrane. Originally identified two decades ago in budding yeast, investigations using several different eukaryotic systems have since made great progress toward determination of the overall structure and organization of the eight exocyst subunits. Studies point to a critical role for the complex as a spatiotemporal regulator through the numerous protein and lipid interactions of its subunits, although a molecular understanding of exocyst function has been challenging to elucidate. Recent progress demonstrates that the exocyst is also important for additional trafficking steps and cellular processes beyond exocytosis, with links to development and disease. In this review, we discuss current knowledge of exocyst architecture, assembly, regulation and its roles in a variety of cellular trafficking pathways.
doi:10.1111/j.1600-0854.2012.01353.x
PMCID: PMC3374049  PMID: 22420621
3.  Myosin V transports secretory vesicles via a Rab GTPase cascade and interaction with the exocyst complex 
Developmental cell  2011;21(6):1156-1170.
Summary
Vesicle transport requires four steps; vesicle formation, movement, tethering and fusion. In yeast, two Rab GTPases, Ypt31/32 are required for post-Golgi vesicle formation. A third Rab GTPase, Sec4, and the exocyst act in tethering and fusion of these vesicles. Vesicle production is coupled to transport via direct interaction between Ypt31/32 and the yeast myosin V, Myo2. Here we show that Myo2 interacts directly with Sec4, and the exocyst subunit Sec15. Disruption of these interactions results in compromised growth and the accumulation of secretory vesicles. We identified the Sec15-binding region on Myo2, and also identified residues on Sec15 required for interaction with Myo2. That Myo2 interacts with Sec15 uncovers additional roles for the exocyst as an adaptor for molecular motors, and implies similar roles for structurally related tethering complexes. Moreover, these studies predict that for many pathways, molecular motors attach to vesicles prior to their formation, and remain attached until fusion.
doi:10.1016/j.devcel.2011.10.009
PMCID: PMC3241923  PMID: 22172676
4.  Show me the MUN-y 
Structure (London, England : 1993)  2011;19(10):1348-1349.
The structure of the MUN domain of the synaptic protein Munc13-1 by Li, et al., in this issue of Structure (Li et al., 2011) shows that seemingly disparate regulators of SNARE-mediated membrane fusion are highly conserved at the structural level.
doi:10.1016/j.str.2011.09.004
PMCID: PMC3202169  PMID: 22000505
5.  Regulation of exocytosis by the exocyst subunit Sec6 and the SM protein Sec1 
Molecular Biology of the Cell  2012;23(2):337-346.
The Sec6 subunit of the multisubunit exocyst tethering complex interacts with the Sec1/Munc18 protein Sec1 and with the t-SNARE Sec9. Assembly of the exocyst upon vesicle arrival at sites of secretion is proposed to release Sec9 for SNARE complex assembly and to recruit Sec1 for interaction with SNARE complexes to facilitate fusion.
Trafficking of protein and lipid cargo through the secretory pathway in eukaryotic cells is mediated by membrane-bound vesicles. Secretory vesicle targeting and fusion require a conserved multisubunit protein complex termed the exocyst, which has been implicated in specific tethering of vesicles to sites of polarized exocytosis. The exocyst is directly involved in regulating soluble N-ethylmaleimide–sensitive factor (NSF) attachment protein receptor (SNARE) complexes and membrane fusion through interactions between the Sec6 subunit and the plasma membrane SNARE protein Sec9. Here we show another facet of Sec6 function—it directly binds Sec1, another SNARE regulator, but of the Sec1/Munc18 family. The Sec6–Sec1 interaction is exclusive of Sec6–Sec9 but compatible with Sec6–exocyst assembly. In contrast, the Sec6–exocyst interaction is incompatible with Sec6–Sec9. Therefore, upon vesicle arrival, Sec6 is proposed to release Sec9 in favor of Sec6–exocyst assembly and to simultaneously recruit Sec1 to sites of secretion for coordinated SNARE complex formation and membrane fusion.
doi:10.1091/mbc.E11-08-0670
PMCID: PMC3258177  PMID: 22114349
6.  A Cytosolic ATM/NEMO/RIP1 Complex Recruits TAK1 To Mediate the NF-κB and p38 Mitogen-Activated Protein Kinase (MAPK)/MAPK-Activated Protein 2 Responses to DNA Damage▿ 
Molecular and Cellular Biology  2011;31(14):2774-2786.
In multiple tumor types, activation of the transcription factor NF-κB increases the resistance of tumor cells to anticancer therapies and contributes to tumor progression. Genotoxic stress induced by chemotherapy or radiation therapy triggers the ATM-dependent translocation of NF-κB essential modifier (NEMO), also designated IκB kinase γ (IKKγ), from the nucleus to the cytosol, resulting in IκB kinase activation by mechanisms not yet fully understood. RIP1 has been implicated in this response and found to be modified in cells with damaged DNA; however, the nature of the RIP1 modification and its precise role in the pathway remain unclear. Here, we show that DNA damage stimulates the formation of a cytosolic complex containing ATM, NEMO (IKKγ), RIP1, and TAK1. We find that RIP1 is modified by SUMO-1 and ubiquitin in response to DNA damage and demonstrate that modified RIP1 is required for NF-κB activation and tumor cell survival. We show that ATM activates TAK1 in a manner dependent on RIP1 and NEMO. We also reveal TAK1 as a central mediator of the alternative DNA damage response pathway mediated by the p38 mitogen-activated protein kinase (MAPK)/MAPK-activated protein 2 (MAPKAP-2) kinases. These findings have translational implications and reveal RIP1 and TAK1 as potential therapeutic targets in chemoresistance.
doi:10.1128/MCB.01139-10
PMCID: PMC3133388  PMID: 21606198
7.  Getting high on traffic 
Cellular Logistics  2011;1(1):41-44.
Molecular mechanisms of secretory and endocytic transport pathways were the focus of a recent ASBMB Special Symposium on Biochemistry of Membrane Traffic, held last October in the picturesque high-altitude setting of Lake Tahoe. An exciting and diverse array of research and discussion topics focused on recent advances in understanding the molecular basis of movement of protein and membrane through the eukaryotic secretory pathway. Highlights included discussions of the common structural and functional features of tethering complexes, the roles of the lipid PI 4P in the late secretory pathway, organization of specific ER sub-domains and a new look at Golgi biogenesis.
doi:10.4161/cl.1.1.14466
PMCID: PMC3109462
meeting report; intracellular traffic; organelle dynamics; membrane dynamics; Lake Tahoe
8.  The structure of the Myo4p globular tail and its function in ASH1 mRNA localization 
The Journal of Cell Biology  2010;189(3):497-510.
A conserved patch of amino acids in the globular tail of type V myosin binds She3p to localize ASH1 mRNA to the bud of dividing yeast cells.
Type V myosin (MyoV)–dependent transport of cargo is an essential process in eukaryotes. Studies on yeast and vertebrate MyoV showed that their globular tails mediate binding to the cargo complexes. In Saccharomyces cerevisiae, the MyoV motor Myo4p interacts with She3p to localize asymmetric synthesis of HO 1 (ASH1) mRNA into the bud of dividing cells. A recent study showed that localization of GFP-MS2–tethered ASH1 particles does not require the Myo4p globular tail, challenging the supposed role of this domain. We assessed ASH1 mRNA and Myo4p distribution more directly and found that their localization is impaired in cells expressing globular tail–lacking Myo4p. In vitro studies further show that the globular tail together with a more N-terminal linker region is required for efficient She3p binding. We also determined the x-ray structure of the Myo4p globular tail and identify a conserved surface patch important for She3p binding. The structure shows pronounced similarities to membrane-tethering complexes and indicates that Myo4p may not undergo auto-inhibition of its motor domain.
doi:10.1083/jcb.201002076
PMCID: PMC2867299  PMID: 20439999
9.  Watching proteins in motion 
Genome Biology  2009;10(10):316.
A report of the 23rd Protein Symposium 'Proteins in Motion', Boston, USA, 23-27 July 2009.
A report of the 23rd Protein Symposium 'Proteins in Motion', Boston, USA, 23-27 July 2009.
doi:10.1186/gb-2009-10-10-316
PMCID: PMC2784317  PMID: 19863776
10.  Crystal structure of the APOBEC3G catalytic domain reveals potential oligomerization interfaces 
Summary
APOBEC3G is a DNA cytidine deaminase that has anti-viral activity against HIV-1 and other pathogenic viruses. In this study the crystal structure of the catalytically active C-terminal domain was determined to 2.25 Å. This structure corroborates features previously observed in NMR studies, a bulge in the second β-strand and a lengthening of the second α-helix. Oligomerization is postulated to be critical for the function of APOBEC3G. In this structure, four extensive intermolecular interfaces are observed suggesting potential models for APOBEC3G oligomerization. The structural and functional significance of these interfaces was probed by solution NMR and disruptive variants were designed and tested for DNA deaminase and anti-HIV activities. The variant designed to disrupt the most extensive interface lost both activities. NMR solution data provides evidence that another interface, which coordinates a novel zinc site, also exists. Thus, the observed crystallographic interfaces of APOBEC3G may be important for both oligomerization and function.
doi:10.1016/j.str.2009.10.016
PMCID: PMC2913127  PMID: 20152150
11.  Sec6p Anchors the Assembled Exocyst Complex at Sites of Secretion 
Molecular Biology of the Cell  2009;20(3):973-982.
The exocyst is an essential protein complex required for targeting and fusion of secretory vesicles to sites of exocytosis at the plasma membrane. To study the function of the exocyst complex, we performed a structure-based mutational analysis of the Saccharomyces cerevisiae exocyst subunit Sec6p. Two “patches” of highly conserved residues are present on the surface of Sec6p; mutation of either patch does not compromise protein stability. Nevertheless, replacement of SEC6 with the patch mutants results in severe temperature-sensitive growth and secretion defects. At nonpermissive conditions, although trafficking of secretory vesicles to the plasma membrane is unimpaired, none of the exocyst subunits are polarized. This is consistent with data from other exocyst temperature-sensitive mutants, which disrupt the integrity of the complex. Surprisingly, however, these patch mutations result in mislocalized exocyst complexes that remain intact. Our results indicate that assembly and polarization of the exocyst are functionally separable events, and that Sec6p is required to anchor exocyst complexes at sites of secretion.
doi:10.1091/mbc.E08-09-0968
PMCID: PMC2633393  PMID: 19073882
12.  Conservation of Helical Bundle Structure between the Exocyst Subunits 
PLoS ONE  2009;4(2):e4443.
Background
The exocyst is a large hetero-octomeric protein complex required for regulating the targeting and fusion of secretory vesicles to the plasma membrane in eukaryotic cells. Although the sequence identity between the eight different exocyst subunits is less than 10%, structures of domains of four of the subunits revealed a similar helical bundle topology. Characterization of several of these subunits has been hindered by lack of soluble protein for biochemical and structural studies.
Methodology/Principal Findings
Using advanced hidden Markov models combined with secondary structure predictions, we detect significant sequence similarity between each of the exocyst subunits, indicating that they all contain helical bundle structures. We corroborate these remote homology predictions by identifying and purifying a predicted domain of yeast Sec10p, a previously insoluble exocyst subunit. This domain is soluble and folded with approximately 60% α-helicity, in agreement with our predictions, and capable of interacting with several known Sec10p binding partners.
Conclusions/Significance
Although all eight of the exocyst subunits had been suggested to be composed of similar helical bundles, this has now been validated by our hidden Markov model structure predictions. In addition, these predictions identified protein domains within the exocyst subunits, resulting in creation and characterization of a soluble, folded domain of Sec10p.
doi:10.1371/journal.pone.0004443
PMCID: PMC2635961  PMID: 19214222
13.  Sec1p Binds to Snare Complexes and Concentrates at Sites of Secretion 
The Journal of Cell Biology  1999;146(2):333-344.
Proteins of the Sec1 family have been shown to interact with target-membrane t-SNAREs that are homologous to the neuronal protein syntaxin. We demonstrate that yeast Sec1p coprecipitates not only the syntaxin homologue Ssop, but also the other two exocytic SNAREs (Sec9p and Sncp) in amounts and in proportions characteristic of SNARE complexes in yeast lysates. The interaction between Sec1p and Ssop is limited by the abundance of SNARE complexes present in sec mutants that are defective in either SNARE complex assembly or disassembly. Furthermore, the localization of green fluorescent protein (GFP)-tagged Sec1p coincides with sites of vesicle docking and fusion where SNARE complexes are believed to assemble and function. The proposal that SNARE complexes act as receptors for Sec1p is supported by the mislocalization of GFP-Sec1p in a mutant defective for SNARE complex assembly and by the robust localization of GFP-Sec1p in a mutant that fails to disassemble SNARE complexes. The results presented here place yeast Sec1p at the core of the exocytic fusion machinery, bound to SNARE complexes and localized to sites of secretion.
PMCID: PMC3206579  PMID: 10427089
Sec1 proteins; syntaxin proteins; SNARE complex; secretion; yeast

Results 1-13 (13)