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Striking the Balance between Selectivity and Efficiency: An Integrative Model of Nucleocytoplasmic Transport
Monday, February 13, 2017 1174-Pos Board B242 TRIM Family Proteins in Intracellular Vesicle Trafficking Kristyn Gumpper1, Ki Ho Park1, Mingzhai Sun1, Pei-Hui Lin1, Tao Tan1, Miyuki Nishi2, Hiroshi Takeshima2, Jianjie Ma1. 1 The Ohio State University, Columbus, OH, USA, 2Kyoto University, Kyoto, Japan. The tripartite motif (TRIM) family of over 75 different proteins serves regulative roles in many critical aspects of health and disease including wound healing, immunity, secretion and cancer. Our laboratory has previously shown that MG53 (TRIM72) played an essential role in cell membrane repair via trafficking of intracellular vesicles to the injury site. TRIM50 is the closest homologue to MG53, however, it has dissimilar cellular function and tissue localization. The molecular mechanisms that underlie the different functions for MG53 and TRIM50 are unknown. An earlier study from our group showed that TRIM50 is predominantly expressed in the stomach, and is involved in vesicle trafficking during acid secretion in gastric parietal cells. While we know that MG53-mediated vesicle translocation to membrane injury site is driven by a molecular motor named non-muscle myosin II-A (NM2A), the molecular motor that facilitates TRIM50 movement during acid secretion is unknown. In this study, we used intracellular vesicle tracking to examine how TRIM50-associated vesicles move within the cell, distinguishing between diffusive and directional ballistic motions. We found that gastric carcinoma TMK-1 cells transfected with mCherry-TRIM50 display non-diffusive movements, and treatment with microtubule inhibitors such as nocodazole and colchicine abolished the directed motion of the TRIM50-associated vesicles. To determine the differential function and cytoskeletal associations of MG53 and TRIM50, chimeric proteins were constructed and tested for response to cell membrane injury, preferential lipid binding (e.g. phosphatidylserine for MG53 vs phosphatidylinositol for TRIM50), and subcellular localization. Overall, our data suggest that TRIM50-vesicles are associated with dynein, a microtubule-associated molecular motor, to power vesicle trafficking related to acid secretion in gastric parietal cells. Additionally, the critical regions responsible for the differential functions of MG53 and TRIM50 was not found within the divergent C-terminal PRY/SPRY domain, as we had predicted. This study suggests that the N-terminal domain of TRIM family proteins may be responsible for vesicle trafficking, providing a clue on how TRIM family proteins regulate vesicle trafficking and how the genetic ablation of TRIM50 is involved in the gastrointestinal symptoms observed in patients with Williams Syndrome. Additionally, it suggests that we could use a shorter form of MG53 as a new therapeutic agent for muscular dystrophy and cardiovascular disease. 1175-Pos Board B243 Membrane Binding and Remodelling by the COPII Coat Sebastian Daum, Mona Grimmer, Jan Ebenhan, Annette Meister, Jan Auerswald, Daniela Kruger, Stefan Werner, Kirsten Bacia. Dept. of Chemistry / Biophysical Chemistry, University of Halle, Halle, Germany. Dynamic remodelling of cellular membranes in conjunction with protein complex assembly and disassembly constitutes a key component of intracellular trafficking pathways. Reconstitution of selected steps outside the cell using artificial membranes facilitates the use of biophysical techniques for investigating structure and dynamics of protein-lipid assemblies. COPIIcoated membrane vesicles are responsible for transporting cargo from the ER towards the Golgi apparatus in the secretory pathway. The small GTPase Sar1, which belongs to the Sar1/Arf family of GTPases and the Rassuperfamily, is an essential component in COPII-vesicle formation. Upon activation by GTP, Sar1 binds to membranes, embedding an amphipathic helix into the proximal leaflet of the bilayer. The role of GTP presence versus GTP hydrolysis in the fission of COPII vesicles is not completely clear. We study COPII vesicle formation in a bottom-up fashion on baker’s yeast proteins and model membranes using cryo-electron microscopy techniques (cryo-EM), confocal fluorescence microscopy and fluorescence correlation spectroscopy (FCS). Cryo-EM shows strongly different COPII coated vesicle morphologies under GTP hydrolyzing versus non-hydrolyzing conditions. More subtle differences are observed by cryo-EM among reconstituted COPII samples under non-hydrolyzing conditions. Membrane binding of the small GTPases Sar1 and Arf1 is studied with biophysical techniques: By combining FCS with a Langmuir film balance, the protein’s footprint in the proximal membrane leaflet is revealed. Dual-color fluorescence cross-correlation spectroscopy (FCCS) analysis has been developed into a method for obtaining binding curves and testing binding models of protein binding to freely diffusing vesicles. The higher specificity makes
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fluorescence cross-correlation advantageous compared to previous fluorescence autocorrelation analysis also for small vesicle binding studies. 1176-Pos Board B244 Striking the Balance between Selectivity and Efficiency: An Integrative Model of Nucleocytoplasmic Transport Barak Raveh1, Jerome Karp2, Samuel Sparks2, Benjamin Timney3, David Cowburn2, Michael P. Rout3, Andrej Sali1. 1 University of California, San Francisco, San Francisco, CA, USA, 2Albert Einstein College of Medicine, New York, NY, USA, 3Rockefeller University, New York, NY, USA. Macromolecular traffic through the nuclear pore complex (NPC) occurs by either passive or facilitated diffusion. While passive diffusion rates are believed to decrease dramatically beyond the approximately 30-60 kDa size threshold, facilitated diffusion, which is mediated by soluble nuclear transport receptors (NTRs), proceeds rapidly even for large macromolecular assemblies such as intact ribosomal subunits. To study the mechanism by which the NPC facilitates rapid facilitated diffusion while maintaining an effective barrier to the passive diffusion of large macromolecules, we created a model of transport dynamics that integrates data from multiple empirical and theoretical sources: small-angle x-ray scattering, nuclear magnetic resonance spectroscopy, electron tomography, dynamic light scattering, atomistic simulations, and additional datasets available from the literature. In both our model and in independent fluorescence recovery after photobleaching (FRAP) measurements, we show that the barrier to passive diffusion is much softer than previously thought, and completely lacks a well-defined size cutoff at 30-60 kDa. Coarse-grained Brownian Dynamics simulations replicate these results, and indicate that this gradual decrease in passive diffusion rates with molecular weight is consistent with an entropic gating mechanism, in which repulsive forces are set out by the crowded and highly dynamic FG repeat domains that fill the central channel of the NPC. In facilitated diffusion, we show that cargo-bound NTRs counter these repulsive forces by utilizing a unique dynamic interaction mechanism with the FG repeats, transitioning between weakly- and strongly-interacting states, thereby sliding and exchanging between proximal FG repeats. This ‘slide-and-exchange’ interaction mechanism is reminiscent of the sliding of transcription factors on DNA molecules. Our results highlight the direct role of the disorder within FG repeats in nucleocytoplasmic transport, and resolve the apparent conflict between the selectivity and speed of transport. 1177-Pos Board B245 Bacterial Carboxysome Shell Proteins are Selectively Permeable to Carbon Fixation Substrates Paween Mahinthichaichan1, Dylan Morris2, Yi Wang3, Grant J. Jensen2, Emad Tajkhorshid1. 1 Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL, USA, 2California Institute of Technology, Pasadena, CA, USA, 3Physics, Chinese University of Hong Kong, Shatin, N.T., Hong Kong. Carboxysome provides an efficient mechanism for cyanobacteria and chemoautotrophs to fix carbon dioxide (CO2) and organic metabolites into building blocks of biomolecules. It is a polyhedral body that encapsulates ribulose1,5-bisphosphate carboxylase/oxygenase (RuBisCO), an enzyme that carries out the CO2 fixation and meanwhile competitively reacts with O2. Its outer surface is coated by the assembly of thousands of small shell proteins. Many of these shell proteins form oligomeric structures with a semi-permeable ˚ radius central pore, suggesting a favorable feature for the binding of 2-3A anions such as bicarbonate (HCO3-), the aqueous soluble form of CO2. The present study examines the translocation HCO3-, CO2 and O2 through the central pores of different isoforms of shell protein complexes from alpha and beta cyanobacteria. We employed umbrella sampling simulations to calculate partitioning free energy profiles of these small molecules and performed detailed electrostatic analysis of the structures. The results demonstrate that carboxysome shell protein oligomers share a unique feature in that their central pores are preferably selective for HCO3- over hydrophobic CO2 and O2 gases. The pores are found to be hydrated and are electropositive, thus favoring HCO3- and Cl- anions. Hence, as the carboxysome encapsulates carbonic anhydrase enzymes which dehydrates HCO3- to CO2, the preferential uptake of HCO3- (instead of CO2) into the carboxysomal lumen from the cytoplasm is an intrinsic way to minimize the reaction between O2 and the RuBisCO and to increase the concentration of CO2 around the RuBisCO. The results also provide insight into the assembled orientations of the shell proteins, which is necessary for solving a full atomic model of the carboxysome.
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fluorescence cross-correlation advantageous compared to previous fluorescence autocorrelation analysis also for small vesicle binding studies. 1176-Pos Board B244 Striking the Balance between Selectivity and Efficiency: An Integrative Model of Nucleocytoplasmic Transport Barak Raveh1, Jerome Karp2, Samuel Sparks2, Benjamin Timney3, David Cowburn2, Michael P. Rout3, Andrej Sali1. 1 University of California, San Francisco, San Francisco, CA, USA, 2Albert Einstein College of Medicine, New York, NY, USA, 3Rockefeller University, New York, NY, USA. Macromolecular traffic through the nuclear pore complex (NPC) occurs by either passive or facilitated diffusion. While passive diffusion rates are believed to decrease dramatically beyond the approximately 30-60 kDa size threshold, facilitated diffusion, which is mediated by soluble nuclear transport receptors (NTRs), proceeds rapidly even for large macromolecular assemblies such as intact ribosomal subunits. To study the mechanism by which the NPC facilitates rapid facilitated diffusion while maintaining an effective barrier to the passive diffusion of large macromolecules, we created a model of transport dynamics that integrates data from multiple empirical and theoretical sources: small-angle x-ray scattering, nuclear magnetic resonance spectroscopy, electron tomography, dynamic light scattering, atomistic simulations, and additional datasets available from the literature. In both our model and in independent fluorescence recovery after photobleaching (FRAP) measurements, we show that the barrier to passive diffusion is much softer than previously thought, and completely lacks a well-defined size cutoff at 30-60 kDa. Coarse-grained Brownian Dynamics simulations replicate these results, and indicate that this gradual decrease in passive diffusion rates with molecular weight is consistent with an entropic gating mechanism, in which repulsive forces are set out by the crowded and highly dynamic FG repeat domains that fill the central channel of the NPC. In facilitated diffusion, we show that cargo-bound NTRs counter these repulsive forces by utilizing a unique dynamic interaction mechanism with the FG repeats, transitioning between weakly- and strongly-interacting states, thereby sliding and exchanging between proximal FG repeats. This ‘slide-and-exchange’ interaction mechanism is reminiscent of the sliding of transcription factors on DNA molecules. Our results highlight the direct role of the disorder within FG repeats in nucleocytoplasmic transport, and resolve the apparent conflict between the selectivity and speed of transport. 1177-Pos Board B245 Bacterial Carboxysome Shell Proteins are Selectively Permeable to Carbon Fixation Substrates Paween Mahinthichaichan1, Dylan Morris2, Yi Wang3, Grant J. Jensen2, Emad Tajkhorshid1. 1 Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL, USA, 2California Institute of Technology, Pasadena, CA, USA, 3Physics, Chinese University of Hong Kong, Shatin, N.T., Hong Kong. Carboxysome provides an efficient mechanism for cyanobacteria and chemoautotrophs to fix carbon dioxide (CO2) and organic metabolites into building blocks of biomolecules. It is a polyhedral body that encapsulates ribulose1,5-bisphosphate carboxylase/oxygenase (RuBisCO), an enzyme that carries out the CO2 fixation and meanwhile competitively reacts with O2. Its outer surface is coated by the assembly of thousands of small shell proteins. Many of these shell proteins form oligomeric structures with a semi-permeable ˚ radius central pore, suggesting a favorable feature for the binding of 2-3A anions such as bicarbonate (HCO3-), the aqueous soluble form of CO2. The present study examines the translocation HCO3-, CO2 and O2 through the central pores of different isoforms of shell protein complexes from alpha and beta cyanobacteria. We employed umbrella sampling simulations to calculate partitioning free energy profiles of these small molecules and performed detailed electrostatic analysis of the structures. The results demonstrate that carboxysome shell protein oligomers share a unique feature in that their central pores are preferably selective for HCO3- over hydrophobic CO2 and O2 gases. The pores are found to be hydrated and are electropositive, thus favoring HCO3- and Cl- anions. Hence, as the carboxysome encapsulates carbonic anhydrase enzymes which dehydrates HCO3- to CO2, the preferential uptake of HCO3- (instead of CO2) into the carboxysomal lumen from the cytoplasm is an intrinsic way to minimize the reaction between O2 and the RuBisCO and to increase the concentration of CO2 around the RuBisCO. The results also provide insight into the assembled orientations of the shell proteins, which is necessary for solving a full atomic model of the carboxysome.