Paul LaPointe

Hsp90 Cochaperone Aha1 Downregulation Rescues Misfolding of CFTR in Cystic Fibrosis

The pathways that distinguish transport of folded and misfolded cargo through the exocytic (secretory) pathway of eukaryotic cells remain unknown. Using proteomics to assess global cystic fibrosis (CF) transmembrane conductance regulator (CFTR) protein interactions (the CFTR interactome), we show that Hsp90 cochaperones modulate Hsp90-dependent stability of CFTR protein folding in the endoplasmic reticulum (ER). Cell-surface rescue of the most common disease variant that is restricted to the ER, DeltaF508, can be initiated by partial siRNA silencing of the Hsp90 cochaperone ATPase regulator Aha1. We propose that failure of DeltaF508 to achieve an energetically favorable fold in response to the steady-state dynamics of the chaperone folding environment (the chaperome) is responsible for the pathophysiology of CF. The activity of cargo-associated chaperome components may be a common mechanism regulating folding for ER exit, providing a general framework for correction of misfolding disease.

Structure of the Sec13/31 COPII coat cage

Endomembranes of eukaryotic cells are dynamic structures that are in continuous communication through the activity of specialized cellular machineries, such as the coat protein complex II (COPII), which mediates cargo export from the endoplasmic reticulum (ER). COPII consists of the Sar1 GTPase, Sec23 and Sec24 (Sec23/24), where Sec23 is a Sar1-specific GTPase-activating protein and Sec24 functions in cargo selection, and Sec13 and Sec31 (Sec13/31), which has a structural role. Whereas recent results have shown that Sec23/24 and Sec13/31 can self-assemble to form COPII cage-like particles, we now show that Sec13/31 can self-assemble to form minimal cages in the absence of Sec23/24. We present a three-dimensional reconstruction of these Sec13/31 cages at 30u2009Å resolution using cryo-electron microscopy and single particle analysis. These results reveal a novel cuboctahedron geometry with the potential to form a flexible lattice and to generate a diverse range of containers. Our data are consistent with a model for COPII coat complex assembly in which Sec23/24 has a non-structural role as a multivalent ligand localizing the self-assembly of Sec13/31 to form a cage lattice driving ER cargo export.

Structural basis for cargo regulation of COPII coat assembly.

Using cryo-electron microscopy, we have solved the structure of an icosidodecahedral COPII coat involved in cargo export from the endoplasmic reticulum (ER) coassembled from purified cargo adaptor Sec23-24 and Sec13-31 lattice-forming complexes. The coat structure shows a tetrameric assembly of the Sec23-24 adaptor layer that is well positioned beneath the vertices and edges of the Sec13-31 lattice. Fitting the known crystal structures of the COPII proteins into the density map reveals a flexible hinge region stemming from interactions between WD40 beta-propeller domains present in Sec13 and Sec31 at the vertices. The structure shows that the hinge region can direct geometric cage expansion to accommodate a wide range of bulky cargo, including procollagen and chylomicrons, that is sensitive to adaptor function in inherited disease. The COPII coat structure leads us to propose a mechanism by which cargo drives cage assembly and membrane curvature for budding from the ER.

The COPII cage: unifying principles of vesicle coat assembly

Communication between compartments of the exocytic and endocytic pathways in eukaryotic cells involves transport carriers — vesicles and tubules — that mediate the vectorial movement of cargo. Recent studies of transport-carrier formation in the early secretory pathway have provided new insights into the mechanisms of cargo selection by coat protein complex-II (COPII) adaptor proteins, the construction of cage-protein scaffolds and fission. These studies are beginning to produce a unifying molecular and structural model of coat function in the formation and fission of vesicles and tubules in endomembrane traffic.

Biological and Structural Basis for Aha1 Regulation of Hsp90 ATPase Activity in Maintaining Proteostasis in the Human Disease Cystic Fibrosis

We propose a general model for the role of the Hsp90 ATPase cycle in proteostasis in which Aha1 regulates the dwell time of Hsp90 with client by integrating chaperone function and client folding energetics by modulating ATPase sensitive N-terminal dimer structural transitions.

Mise en Place–This Bud's for the Golgi

Selective cargo export from the endoplasmic reticulum is brought about by the budding of COPII vesicles. While the main structural components of the COPII coat have been identified and characterized, the regulatory event(s) promoting COPII vesicle biogenesis and cargo selection still remains largely unknown. New data by Glick and colleagues suggest that Sec12 and COPII function may be downstream of important early events coordinated by transitional ER (tER) exit sites.

Microsome-Based Assay for Analysis of Endoplasmic Reticulum to Golgi Transport in Mammalian Cells

Publisher Summary The trafficking of proteins along the first stage of the secretory pathway is mediated by small vesicles that bud from the endoplasmic reticulum (ER) and subsequently fuse with the cis-Golgi compartment. To follow vesicle formation, a differential centrifugation procedure is employed to separate the more rapidly sedimenting ER and Golgi membranes from the slowly sedimenting vesicles. Consumption is analyzed using a two-stage assay in which vesicles isolated by differential centrifugation during stage 1 are subsequently added to stage 2 (fusion) reactions containing acceptor Golgi membranes. Use a pipette to transfer the cells to 50-ml plastic tubes and then repeat the scraping procedure to ensure that all the cells are collected. Centrifuge at 720xg for 3 min and remove the supernatant by aspiration. Resuspend each cell pellet in 0.9 ml of homogenization buffer supplemented with PIC and homogenize by three complete passes through a 1-ml ball-bearing homogenizer. Remove the supernatants by aspiration, wash the pellets with 2 ml of transport buffer, and combine the membranes into two 1.5-ml microfuge tubes.

Applications of Automated Electron Microscopy: Using Leginon to Study the Structure of COPII Protein Complexes

COPII proteins are responsible for forming the vesicles that transport proteins from the endoplasmic reticulum to the Golgi apparatus. The COPII proteins form a coat around the budding vesicle and are responsible for both selecting the protein cargo and drawing the ER membrane up and pinching it into a vesicle. COPII coats consist of three components: Sar1, a GTPase; Sec23/24, a GTPase activating protein (GAP); and Sec13/31, a GAP stimulator which has also been implicated in inducing membrane curvature. While structures of Sar1 and Sec23/24 have been solved, little is known about the structure of Sec13/31 or the structure of the COPII lattice. We are investigating the structure of the COPII lattice using cryo-electron microscopy (cryoEM).

Purification and Properties of Mammalian Sec23/24 from Insect Cells

The Sec23/24 complex is a large heterodimeric protein involved in COPII vesicle biogenesis. The individual mammalian protein subunits are too large for expression in bacterial systems. This article details the use of the Bac-to-Bac baculovirus coexpression system in insect cells for both the human Sec23A and Sec24C. This strategy results in high yields of pure, functional protein and can be adapted for the purification of other Sec23/24 isoforms for their biochemical and biological characterization.