To protect or reject

A protein known for its role in dismantling faulty SNARE complexes can also help to maintain complexes that have formed properly during membrane fusion

Watching Proteins in Motion

A report of the 23rd Protein Symposium \u27Proteins in Motion\u27, Boston, USA, 23-27 July 2009

The Secret Life of Tethers: The Role of Tethering Factors in SNARE Complex Regulation

Trafficking in eukaryotic cells is a tightly regulated process to ensure correct cargo delivery to the proper destination organelle or plasma membrane. In this review, we focus on how the vesicle fusion machinery, the SNARE complex, is regulated by the interplay of the multisubunit tethering complexes (MTC) with the SNAREs and Sec1/Munc18 (SM) proteins. Although these factors are used in different stages of membrane trafficking, e.g., Golgi to plasma membrane transport vs. vacuolar fusion, and in a variety of diverse eukaryotic cell types, many commonalities between their functions are being revealed. We explore the various protein-protein interactions and findings from functional reconstitution studies in order to highlight both their common features and the differences in their modes of regulation. These studies serve as a starting point for mechanistic explorations in other systems

Expansion of Comprehensive Screening of Male Sexually Transmitted Infection Clinic Attendees with \u3cem\u3eMycoplasma genitalium\u3c/em\u3e and \u3cem\u3eTrichomonas vaginalis\u3c/em\u3e Molecular Assessment: a Retrospective Analysis

Of 1,493 encounters of males at a sexually transmitted infection (STI) clinic in a community with a high prevalence of STI, Chlamydia trachomatis was detected in 8.7% and Neisseria gonorrhoeae was detected in 6.6%. Additional Trichomonas vaginalis and Mycoplasma genitalium screening found 17.4% and 23.9% of the encounters, respectively, to be positive for STI. STI agents were detected in 13.7% of urine specimens; addition of pharyngeal and rectal collections to the analysis resulted in detection of STI agents in 19.0% and 23.9% of encounters, respectively. A total of 101 (23.8%) encounters of identified STI involved sole detection of M. genitalium. Expansion of the STI analyte panel (including M. genitalium) and additional specimen source sampling within a comprehensive STI screening program increase identification of male STI carriers

Speeding up protein folding: mutations that increase the rate at which Rop folds and unfolds by over four orders of magnitude

BackgroundThe dimeric four-helix-bundle protein Rop folds and unfolds extremely slowly. To understand the molecular basis for the slow kinetics, we have studied the folding and unfolding of wild-type Rop and a series of hydrophobic core mutants.ResultsMutation of the hydrophobic core creates stable, dimeric, and wild-type-like proteins with dramatically increased rates of both folding and unfolding. The increases in rates are dependent upon the number and position of repacked residues within the hydrophobic core.ConclusionRop folds by a rapid collision of monomers to form a dimeric intermediate with substantial helical content, followed by a slow rearrangement to the final native structure. Rop unfolding is a single extremely slow kinetic phase. The slow steps of both folding and unfolding are dramatically increased by hydrophobic core replacements, suggesting that their main effect is to substantially decrease the energy of the transition state

The Exocyst Subunit Sec6 Interacts with Assembled Exocytic SNARE Complexes

In eukaryotic cells, membrane-bound vesicles carry cargo between intracellular compartments, to and from the cell surface, and into the extracellular environment. Many conserved families of proteins are required for properly localized vesicle fusion, including the multisubunit tethering complexes and the SNARE complexes. These protein complexes work together to promote proper vesicle fusion in intracellular trafficking pathways. However, the mechanism by which the exocyst, the exocytosis-specific multisubunit tethering complex, interacts with the exocytic SNAREs to mediate vesicle targeting and fusion is currently unknown. We have demonstrated previously that the Saccharomyces cerevisiae exocyst subunit Sec6 directly bound the plasma membrane SNARE protein Sec9 in vitro and that Sec6 inhibited the assembly of the binary Sso1-Sec9 SNARE complex. Therefore, we hypothesized that the interaction between Sec6 and Sec9 prevented the assembly of premature SNARE complexes at sites of exocytosis. To map the determinants of this interaction, we used cross-linking and mass spectrometry analyses to identify residues required for binding. Mutation of residues identified by this approach resulted in a growth defect when introduced into yeast. Contrary to our previous hypothesis, we discovered that Sec6 does not change the rate of SNARE assembly but, rather, binds both the binary Sec9-Sso1 and ternary Sec9-Sso1-Snc2 SNARE complexes. Together, these results suggest a new model in which Sec6 promotes SNARE complex assembly, similar to the role proposed for other tether subunit-SNARE interactions

Conservation of Helical Bundle Structure between the Exocyst Subunits

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 % a-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, folde

Nuclear Export Through Nuclear Envelope Remodeling in Saccharomyces cerevisiae [preprint]

In eukaryotes, subsets of exported mRNAs are organized into large ribonucleoprotein (megaRNP) granules. How megaRNPs exit the nucleus is unclear, as their diameters are much larger than the nuclear pore complex (NPC) central channel. We previously identified a non-canonical nuclear export mechanism in Drosophila (Speese et al., Cell 2012) and mammals (Ding et al., in preparation), in which megaRNPs exit the nucleus by budding across nuclear envelope (NE) membranes. Here, we present evidence for a similar pathway in the nucleus of the budding yeast S. cerevisiae, which contain morphologically similar granules bearing mRNAs. Wild-type yeast displayed these granules at very low frequency, but this frequency was dramatically increased when the non-essential NPC protein Nup116 was deleted. These granules were not artifacts of defective NPCs; a mutation in the exportin XPO1 (CRM1), in which NPCs are normal, induced similar megaRNP upregulation. We hypothesize that a non-canonical nuclear export pathway, analogous to those observed in Drosophila and in mammalian cells, exists in yeast, and that this pathway is upregulated for use when NPCs or nuclear export are impaired

On the brink of change: plant responses to climate on the Colorado Plateau

The intensification of aridity due to anthropogenic climate change in the southwestern U.S. is likely to have a large impact on the growth and survival of plant species that may already be vulnerable to water stress. To make accurate predictions of plant responses to climate change, it is essential to determine the long-term dynamics of plant species associated with past climate conditions. Here we show how the plant species and functional types across a wide range of environmental conditions in Colorado Plateau national parks have changed with climate variability over the last twenty years. During this time, regional mean annual temperature increased by 0.188C per year from 1989–1995, 0.068C per year from 1995–2003, declined by 0.148C from 2003–2008, and there was high interannual variability in precipitation. Non-metric multidimensional scaling of plant species at long-term monitoring sites indicated five distinct plant communities. In many of the communities, canopy cover of perennial plants was sensitive to mean annual temperature occurring in the previous year, whereas canopy cover of annual plants responded to cool season precipitation. In the perennial grasslands, there was an overall decline of C3 perennial grasses, no change of C4 perennial grasses, and an increase of shrubs with increasing temperature. In the shrublands, shrubs generally showed no change or slightly increased with increasing temperature. However, certain shrub species declined where soil and physical characteristics of a site limited water availability. In the higher elevation woodlands, Juniperus osteosperma and shrub canopy cover increased with increasing temperature, while Pinus edulis at the highest elevation sites was unresponsive to interannual temperature variability. These results from well-protected national parks highlight the importance of temperature to plant responses in a water-limited region and suggest that projected increases in aridity are likely to promote grass loss and shrub expansion on the Colorado Plateau