Cryo-EM structure of metazoan TRAPPIII, the multi-subunit complex that activates the GTPase Rab1.

The TRAPP complexes are nucleotide exchange factors that play essential roles in membrane traffic and autophagy. TRAPPII activates Rab11, and TRAPPIII activates Rab1, with the two complexes sharing a core of small subunits that affect nucleotide exchange but being distinguished by specific large subunits that are essential for activity in vivo. Crystal structures of core subunits have revealed the mechanism of Rab activation, but how the core and the large subunits assemble to form the complexes is unknown. We report a cryo-EM structure of the entire Drosophila TRAPPIII complex. The TRAPPIII-specific subunits TRAPPC8 and TRAPPC11 hold the catalytic core like a pair of tongs, with TRAPPC12 and TRAPPC13 positioned at the joint between them. TRAPPC2 and TRAPPC2L link the core to the two large arms, with the interfaces containing residues affected by disease-causing mutations. The TRAPPC8 arm is positioned such that it would contact Rab1 that is bound to the core, indicating how the arm could determine the specificity of the complex. A lower resolution structure of TRAPPII shows a similar architecture and suggests that the TRAPP complexes evolved from a single ur-TRAPP

Pressure and Chemical Unfolding of an α-Helical Bundle Protein: The GH2 Domain of the Protein Adaptor GIPC1.

When combined with NMR spectroscopy, high hydrostatic pressure is an alternative perturbation method used to destabilize globular proteins that has proven to be particularly well suited for exploring the unfolding energy landscape of small single-domain proteins. To date, investigations of the unfolding landscape of all-β or mixed-α/β protein scaffolds are well documented, whereas such data are lacking for all-α protein domains. Here we report the NMR study of the unfolding pathways of GIPC1-GH2, a small α-helical bundle domain made of four antiparallel α-helices. High-pressure perturbation was combined with NMR spectroscopy to unravel the unfolding landscape at three different temperatures. The results were compared to those obtained from classical chemical denaturation. Whatever the perturbation used, the loss of secondary and tertiary contacts within the protein scaffold is almost simultaneous. The unfolding transition appeared very cooperative when using high pressure at high temperature, as was the case for chemical denaturation, whereas it was found more progressive at low temperature, suggesting the existence of a complex folding pathway

Cryo-EM structure of metazoan TRAPPIII, the multi-subunit complex that activates the GTPase Rab1.

The TRAPP complexes are nucleotide exchange factors that play essential roles in membrane traffic and autophagy. TRAPPII activates Rab11, and TRAPPIII activates Rab1, with the two complexes sharing a core of small subunits that affect nucleotide exchange but being distinguished by specific large subunits that are essential for activity in vivo. Crystal structures of core subunits have revealed the mechanism of Rab activation, but how the core and the large subunits assemble to form the complexes is unknown. We report a cryo-EM structure of the entire Drosophila TRAPPIII complex. The TRAPPIII-specific subunits TRAPPC8 and TRAPPC11 hold the catalytic core like a pair of tongs, with TRAPPC12 and TRAPPC13 positioned at the joint between them. TRAPPC2 and TRAPPC2L link the core to the two large arms, with the interfaces containing residues affected by disease-causing mutations. The TRAPPC8 arm is positioned such that it would contact Rab1 that is bound to the core, indicating how the arm could determine the specificity of the complex. A lower resolution structure of TRAPPII shows a similar architecture and suggests that the TRAPP complexes evolved from a single ur-TRAPP

Zasp52 strengthens whole embryo tissue integrity through supracellular actomyosin networks.

Peer reviewed: TrueAcknowledgements: The authors thank Frieder Schöck, Debbie Andrew, Magali Suzanne and Isabel Palacios for fly stocks, Emmanuel Derivery for reagents and members of the lab and Cell Biology division for input on the manuscript.Funder: UK Research and InnovationFunder: MRC Laboratory of Molecular BiologyDuring morphogenesis, large-scale changes of tissue primordia are coordinated across an embryo. In Drosophila, several tissue primordia and embryonic regions are bordered or encircled by supracellular actomyosin cables, junctional actomyosin enrichments networked between many neighbouring cells. We show that the single Drosophila Alp/Enigma-family protein Zasp52, which is most prominently found in Z-discs of muscles, is a component of many supracellular actomyosin structures during embryogenesis, including the ventral midline and the boundary of the salivary gland placode. We reveal that Zasp52 contains within its central coiled-coil region a type of actin-binding motif usually found in CapZbeta proteins, and this domain displays actin-binding activity. Using endogenously-tagged lines, we identify that Zasp52 interacts with junctional components, including APC2, Polychaetoid and Sidekick, and actomyosin regulators. Analysis of zasp52 mutant embryos reveals that the severity of the embryonic defects observed scales inversely with the amount of functional protein left. Large tissue deformations occur where actomyosin cables are found during embryogenesis, and in vivo and in silico analyses suggest a model whereby supracellular Zasp52-containing cables aid to insulate morphogenetic changes from one another

Macromolecular condensation buffers intracellular water potential.

Acknowledgements: The order of the second and corresponding authors is arbitrary and these authors can change the order of their respective names to suit their own interests. This work has been supported by the Medical Research Council, as part of United Kingdom Research and Innovation (MC_UP_1201/13 to E.D.; MC_UP_1201/4 to J.S.O. and MCMB MR/V028669/1 to J.E.C.), the Human Frontier Science Program (Career Development Award CDA00034/2017 to E.D.), a Versus Arthritis Senior Research Fellowship Award (20875 to Q.-J.M.) and an MRC project grant (MR/K019392/1 to Q.-J.M.), a Grifols ‘ALTA’ Alpha-1-Antitrypsin Laurell’s Training Award and an Alpha-1-Foundation (grant number 614939) to J.E.C., and by a Wellcome Trust Sir Henry Dale Fellowship (208790/Z/17/Z to R.S.E.). N.M.R. is supported by a Medical Research Council Clinician Scientist Fellowship (MR/S022023/1). L.K.K. and V.J.P.-H. are recipients of EMBO Postdoctoral fellowships (ALTF 876-2021 and ALTF 577-2018, respectively). K.E.M. is supported by the Wellcome Trust through a Sir Henry Wellcome Postdoctoral Fellowship (220480/Z/20/Z). P.M.M. and J.B. were supported by Volkswagen ‘Life’ grant number 96827 and the DFG Excellence Cluster Physics of Life. We thank H. Andreas for frog maintenance; C. Godlee and M. Kaksonen for the gift of unpublished S. cerevisiae yeast strains and initial discussion of yeast experiments about temperature; P. Tran for S. pombe yeast strains; L. Miller for help with yeast work; A. Bertolotti for the kind gift of SH-SY5Y cells; and C. Russo, F. Jülicher, M. Gonzalez-Gaitan, K. Kruse, L. Blanchoin, J. Löwe, R. Hegde, P. Farrell and P. Crosby for discussion and suggestions; the staff at the companies Cherry Biotech and Elvesys, in particular T. Guérinier, for their help in designing and assembling the custom microfluidics system required for this project; the members of the Electronics and Mechanical workshops of the LMB for key support; the staff at the LMB Mass Spectrometry facility for performing and analysing MS data; and A. Prasad and T. Stevens for sharing the scripts for protein disorder and kinase motif predictions, respectively. Cartoons were created using BioRender. For the purpose of open access, the MRC Laboratory of Molecular Biology has applied a CC BY public copyright licence to any author accepted manuscript version arising.Optimum protein function and biochemical activity critically depends on water availability because solvent thermodynamics drive protein folding and macromolecular interactions1. Reciprocally, macromolecules restrict the movement of 'structured' water molecules within their hydration layers, reducing the available 'free' bulk solvent and therefore the total thermodynamic potential energy of water, or water potential. Here, within concentrated macromolecular solutions such as the cytosol, we found that modest changes in temperature greatly affect the water potential, and are counteracted by opposing changes in osmotic strength. This duality of temperature and osmotic strength enables simple manipulations of solvent thermodynamics to prevent cell death after extreme cold or heat shock. Physiologically, cells must sustain their activity against fluctuating temperature, pressure and osmotic strength, which impact water availability within seconds. Yet, established mechanisms of water homeostasis act over much slower timescales2,3; we therefore postulated the existence of a rapid compensatory response. We find that this function is performed by water potential-driven changes in macromolecular assembly, particularly biomolecular condensation of intrinsically disordered proteins. The formation and dissolution of biomolecular condensates liberates and captures free water, respectively, quickly counteracting thermal or osmotic perturbations of water potential, which is consequently robustly buffered in the cytoplasm. Our results indicate that biomolecular condensation constitutes an intrinsic biophysical feedback response that rapidly compensates for intracellular osmotic and thermal fluctuations. We suggest that preserving water availability within the concentrated cytosol is an overlooked evolutionary driver of protein (dis)order and function

Macromolecular condensation buffers intracellular water potential

Acknowledgements: The order of the second and corresponding authors is arbitrary and these authors can change the order of their respective names to suit their own interests. This work has been supported by the Medical Research Council, as part of United Kingdom Research and Innovation (MC_UP_1201/13 to E.D.; MC_UP_1201/4 to J.S.O. and MCMB MR/V028669/1 to J.E.C.), the Human Frontier Science Program (Career Development Award CDA00034/2017 to E.D.), a Versus Arthritis Senior Research Fellowship Award (20875 to Q.-J.M.) and an MRC project grant (MR/K019392/1 to Q.-J.M.), a Grifols ‘ALTA’ Alpha-1-Antitrypsin Laurell’s Training Award and an Alpha-1-Foundation (grant number 614939) to J.E.C., and by a Wellcome Trust Sir Henry Dale Fellowship (208790/Z/17/Z to R.S.E.). N.M.R. is supported by a Medical Research Council Clinician Scientist Fellowship (MR/S022023/1). L.K.K. and V.J.P.-H. are recipients of EMBO Postdoctoral fellowships (ALTF 876-2021 and ALTF 577-2018, respectively). K.E.M. is supported by the Wellcome Trust through a Sir Henry Wellcome Postdoctoral Fellowship (220480/Z/20/Z). P.M.M. and J.B. were supported by Volkswagen ‘Life’ grant number 96827 and the DFG Excellence Cluster Physics of Life. We thank H. Andreas for frog maintenance; C. Godlee and M. Kaksonen for the gift of unpublished S. cerevisiae yeast strains and initial discussion of yeast experiments about temperature; P. Tran for S. pombe yeast strains; L. Miller for help with yeast work; A. Bertolotti for the kind gift of SH-SY5Y cells; and C. Russo, F. Jülicher, M. Gonzalez-Gaitan, K. Kruse, L. Blanchoin, J. Löwe, R. Hegde, P. Farrell and P. Crosby for discussion and suggestions; the staff at the companies Cherry Biotech and Elvesys, in particular T. Guérinier, for their help in designing and assembling the custom microfluidics system required for this project; the members of the Electronics and Mechanical workshops of the LMB for key support; the staff at the LMB Mass Spectrometry facility for performing and analysing MS data; and A. Prasad and T. Stevens for sharing the scripts for protein disorder and kinase motif predictions, respectively. Cartoons were created using BioRender. For the purpose of open access, the MRC Laboratory of Molecular Biology has applied a CC BY public copyright licence to any author accepted manuscript version arising.Optimum protein function and biochemical activity critically depends on water availability because solvent thermodynamics drive protein folding and macromolecular interactions1. Reciprocally, macromolecules restrict the movement of ‘structured’ water molecules within their hydration layers, reducing the available ‘free’ bulk solvent and therefore the total thermodynamic potential energy of water, or water potential. Here, within concentrated macromolecular solutions such as the cytosol, we found that modest changes in temperature greatly affect the water potential, and are counteracted by opposing changes in osmotic strength. This duality of temperature and osmotic strength enables simple manipulations of solvent thermodynamics to prevent cell death after extreme cold or heat shock. Physiologically, cells must sustain their activity against fluctuating temperature, pressure and osmotic strength, which impact water availability within seconds. Yet, established mechanisms of water homeostasis act over much slower timescales2, 3; we therefore postulated the existence of a rapid compensatory response. We find that this function is performed by water potential-driven changes in macromolecular assembly, particularly biomolecular condensation of intrinsically disordered proteins. The formation and dissolution of biomolecular condensates liberates and captures free water, respectively, quickly counteracting thermal or osmotic perturbations of water potential, which is consequently robustly buffered in the cytoplasm. Our results indicate that biomolecular condensation constitutes an intrinsic biophysical feedback response that rapidly compensates for intracellular osmotic and thermal fluctuations. We suggest that preserving water availability within the concentrated cytosol is an overlooked evolutionary driver of protein (dis)order and function

Macromolecular condensation buffers intracellular water potential

Acknowledgements: The order of the second and corresponding authors is arbitrary and these authors can change the order of their respective names to suit their own interests. This work has been supported by the Medical Research Council, as part of United Kingdom Research and Innovation (MC_UP_1201/13 to E.D.; MC_UP_1201/4 to J.S.O. and MCMB MR/V028669/1 to J.E.C.), the Human Frontier Science Program (Career Development Award CDA00034/2017 to E.D.), a Versus Arthritis Senior Research Fellowship Award (20875 to Q.-J.M.) and an MRC project grant (MR/K019392/1 to Q.-J.M.), a Grifols ‘ALTA’ Alpha-1-Antitrypsin Laurell’s Training Award and an Alpha-1-Foundation (grant number 614939) to J.E.C., and by a Wellcome Trust Sir Henry Dale Fellowship (208790/Z/17/Z to R.S.E.). N.M.R. is supported by a Medical Research Council Clinician Scientist Fellowship (MR/S022023/1). L.K.K. and V.J.P.-H. are recipients of EMBO Postdoctoral fellowships (ALTF 876-2021 and ALTF 577-2018, respectively). K.E.M. is supported by the Wellcome Trust through a Sir Henry Wellcome Postdoctoral Fellowship (220480/Z/20/Z). P.M.M. and J.B. were supported by Volkswagen ‘Life’ grant number 96827 and the DFG Excellence Cluster Physics of Life. We thank H. Andreas for frog maintenance; C. Godlee and M. Kaksonen for the gift of unpublished S. cerevisiae yeast strains and initial discussion of yeast experiments about temperature; P. Tran for S. pombe yeast strains; L. Miller for help with yeast work; A. Bertolotti for the kind gift of SH-SY5Y cells; and C. Russo, F. Jülicher, M. Gonzalez-Gaitan, K. Kruse, L. Blanchoin, J. Löwe, R. Hegde, P. Farrell and P. Crosby for discussion and suggestions; the staff at the companies Cherry Biotech and Elvesys, in particular T. Guérinier, for their help in designing and assembling the custom microfluidics system required for this project; the members of the Electronics and Mechanical workshops of the LMB for key support; the staff at the LMB Mass Spectrometry facility for performing and analysing MS data; and A. Prasad and T. Stevens for sharing the scripts for protein disorder and kinase motif predictions, respectively. Cartoons were created using BioRender. For the purpose of open access, the MRC Laboratory of Molecular Biology has applied a CC BY public copyright licence to any author accepted manuscript version arising.Optimum protein function and biochemical activity critically depends on water availability because solvent thermodynamics drive protein folding and macromolecular interactions1. Reciprocally, macromolecules restrict the movement of ‘structured’ water molecules within their hydration layers, reducing the available ‘free’ bulk solvent and therefore the total thermodynamic potential energy of water, or water potential. Here, within concentrated macromolecular solutions such as the cytosol, we found that modest changes in temperature greatly affect the water potential, and are counteracted by opposing changes in osmotic strength. This duality of temperature and osmotic strength enables simple manipulations of solvent thermodynamics to prevent cell death after extreme cold or heat shock. Physiologically, cells must sustain their activity against fluctuating temperature, pressure and osmotic strength, which impact water availability within seconds. Yet, established mechanisms of water homeostasis act over much slower timescales2, 3; we therefore postulated the existence of a rapid compensatory response. We find that this function is performed by water potential-driven changes in macromolecular assembly, particularly biomolecular condensation of intrinsically disordered proteins. The formation and dissolution of biomolecular condensates liberates and captures free water, respectively, quickly counteracting thermal or osmotic perturbations of water potential, which is consequently robustly buffered in the cytoplasm. Our results indicate that biomolecular condensation constitutes an intrinsic biophysical feedback response that rapidly compensates for intracellular osmotic and thermal fluctuations. We suggest that preserving water availability within the concentrated cytosol is an overlooked evolutionary driver of protein (dis)order and function

Macromolecular condensation buffers intracellular water potential

Optimum protein function and biochemical activity critically depends on water availability because solvent thermodynamics drive protein folding and macromolecular interactions1. Reciprocally, macromolecules restrict the movement of 'structured' water molecules within their hydration layers, reducing the available 'free' bulk solvent and therefore the total thermodynamic potential energy of water, or water potential. Here, within concentrated macromolecular solutions such as the cytosol, we found that modest changes in temperature greatly affect the water potential, and are counteracted by opposing changes in osmotic strength. This duality of temperature and osmotic strength enables simple manipulations of solvent thermodynamics to prevent cell death after extreme cold or heat shock. Physiologically, cells must sustain their activity against fluctuating temperature, pressure and osmotic strength, which impact water availability within seconds. Yet, established mechanisms of water homeostasis act over much slower timescales2,3; we therefore postulated the existence of a rapid compensatory response. We find that this function is performed by water potential-driven changes in macromolecular assembly, particularly biomolecular condensation of intrinsically disordered proteins. The formation and dissolution of biomolecular condensates liberates and captures free water, respectively, quickly counteracting thermal or osmotic perturbations of water potential, which is consequently robustly buffered in the cytoplasm. Our results indicate that biomolecular condensation constitutes an intrinsic biophysical feedback response that rapidly compensates for intracellular osmotic and thermal fluctuations. We suggest that preserving water availability within the concentrated cytosol is an overlooked evolutionary driver of protein (dis)order and function.</p