Mechanism of an intramembrane chaperone for multipass membrane proteins.

Multipass membrane proteins play numerous roles in biology and include receptors, transporters, ion channels and enzymes1,2. How multipass proteins are co-translationally inserted and folded at the endoplasmic reticulum is not well understood2. The prevailing model posits that each transmembrane domain (TMD) of a multipass protein successively passes into the lipid bilayer through a front-side lateral gate of the Sec61 protein translocation channel3-9. The PAT complex, an intramembrane chaperone comprising Asterix and CCDC47, engages early TMDs of multipass proteins to promote their biogenesis by an unknown mechanism10. Here, biochemical and structural analysis of intermediates during multipass protein biogenesis showed that the nascent chain is not engaged with Sec61, which is occluded and latched closed by CCDC47. Instead, Asterix binds to and redirects the substrate to a location behind Sec61, where the PAT complex contributes to a multipass translocon surrounding a semi-enclosed, lipid-filled cavity11. Detection of multiple TMDs in this cavity after their emergence from the ribosome suggests that multipass proteins insert and fold behind Sec61. Accordingly, biogenesis of several multipass proteins was unimpeded by inhibitors of the Sec61 lateral gate. These findings elucidate the mechanism of an intramembrane chaperone and suggest a new framework for multipass membrane protein biogenesis at the endoplasmic reticulum.

Conformational dynamics of the Hsp70 chaperone throughout key steps of its ATPase cycle.

The 70 kDa heat shock proteins (Hsp70s) are highly versatile molecular chaperones that assist in a wide variety of protein-folding processes. They exert their functions by continuously cycling between states of low and high affinity for client polypeptides, driven by ATP-binding and hydrolysis. This cycling is tuned by cochaperones and clients. Although structures for the high and low client affinity conformations of Hsp70 and Hsp70 domains in complex with various cochaperones and peptide clients are available, it is unclear how structural rearrangements in the presence of cochaperones and clients are orchestrated in space and time. Here, we report insights into the conformational dynamics of the prokaryotic model Hsp70 DnaK throughout its adenosine-5'-triphosphate hydrolysis (ATPase) cycle using proximity-induced fluorescence quenching. Our data suggest that ATP and cochaperone-induced structural rearrangements in DnaK occur in a sequential manner and resolve hitherto unpredicted cochaperone and client-induced structural rearrangements. Peptides induce large conformational changes in DnaK·ATP prior to ATP hydrolysis, whereas a protein client induces significantly smaller changes but is much more effective in stimulating ATP hydrolysis. Analysis of the enthalpies of activation for the ATP-induced opening of the DnaK lid in the presence of clients indicates that the lid does not exert an enthalpic pulling force onto bound clients, suggesting entropic pulling as a major mechanism for client unfolding. Our data reveal important insights into the mechanics, allostery, and dynamics of Hsp70 chaperones. We established a methodology for understanding the link between dynamics and function, Hsp70 diversity, and activity modulation.

EMC rectifies the topology of multipass membrane proteins.

Most eukaryotic multipass membrane proteins are inserted into the membrane of the endoplasmic reticulum. Their transmembrane domains (TMDs) are thought to be inserted co-translationally as they emerge from a membrane-bound ribosome. Here we find that TMDs near the carboxyl terminus of mammalian multipass proteins are inserted post-translationally by the endoplasmic reticulum membrane protein complex (EMC). Site-specific crosslinking shows that the EMC's cytosol-facing hydrophilic vestibule is adjacent to a pre-translocated C-terminal tail. EMC-mediated insertion is mostly agnostic to TMD hydrophobicity, favored for short uncharged C-tails and stimulated by a preceding unassembled TMD bundle. Thus, multipass membrane proteins can be released by the ribosome-translocon complex in an incompletely inserted state, requiring a separate EMC-mediated post-translational insertion step to rectify their topology, complete biogenesis and evade quality control. This sequential co-translational and post-translational mechanism may apply to ~250 diverse multipass proteins, including subunits of the pentameric ion channel family that are crucial for neurotransmission.

The Biogenesis of Multipass Membrane Proteins.

Multipass membrane proteins contain two or more α-helical transmembrane domains (TMDs) that span the lipid bilayer. They are inserted cotranslationally into the prokaryotic plasma membrane or eukaryotic endoplasmic reticulum membrane. The Sec61 complex (SecY complex in prokaryotes) provides a ribosome docking site, houses a channel across the membrane, and contains a lateral gate that opens toward the lipid bilayer. Model multipass proteins can be stitched into the membrane by iteratively using Sec61's lateral gate for TMD insertion and its central pore for translocation of flanking domains. Native multipass proteins, with their diverse TMDs and complex topologies, often also rely on members of the Oxa1 family of translocation factors, the PAT complex chaperone, and other poorly understood factors. Here, we discuss the mechanisms of TMD insertion, highlight the limitations of an iterative insertion model, and propose a new hypothesis for multipass membrane protein biogenesis based on recent findings.

A Novel Murine Model of Parvovirus Associated Dilated Cardiomyopathy Induced by Immunization with VP1-Unique Region of Parvovirus B19.

Background. Parvovirus B19 (B19V) is a common finding in endomyocardial biopsy specimens from myocarditis and dilated cardiomyopathy patients. However, current understanding of how B19V is contributing to cardiac damage is rather limited due to the lack of appropriate mice models. In this work we demonstrate that immunization of BALB/c mice with the major immunogenic determinant of B19V located in the unique sequence of capsid protein VP1 (VP1u) is an adequate model to study B19V associated heart damage. Methods and Results. We immunized mice in the experimental group with recombinant VP1u; immunization with cardiac myosin derived peptide served as a positive reference and phosphate buffered saline served as negative control. Cardiac function and dimensions were followed echocardiographically 69 days after immunization. Progressive dilatation of left ventricle and decline of ejection fraction were observed in VP1u- and myosin-immunized mice. Histologically, severe cardiac fibrosis and accumulation of heart failure cells in lungs were observed 69 days after immunization. Transcriptomic profiling revealed ongoing cardiac remodeling and immune process in VP1u- and myosin-immunized mice. Conclusions. Immunization of BALB/c mice with VP1u induces dilated cardiomyopathy in BALB/c mice and it could be used as a model to study clinically relevant B19V associated cardiac damage.