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Membrane proteins with multiple transmembrane domains play critical roles in cell physiology, but little is known about the machinery coordinating their biogenesis at the endoplasmic reticulum. Here we describe a ~ 360 kDa ribosome-associated complex comprising the core Sec61 channel and five accessory factors: TMCO1, CCDC47 and the Nicalin-TMEM147-NOMO complex. Cryo-electron microscopy reveals a large assembly at the ribosome exit tunnel organized around a central membrane cavity. Similar to protein-conducting channels that facilitate movement of transmembrane segments, cytosolic and luminal funnels in TMCO1 and TMEM147, respectively, suggest routes into the central membrane cavity. High-throughput mRNA sequencing shows selective translocon engagement with hundreds of different multi-pass membrane proteins. Consistent with a role in multi-pass membrane protein biogenesis, cells lacking different accessory components show reduced levels of one such client, the glutamate transporter EAAT1. These results identify a new human translocon and provide a molecular framework for understanding its role in multi-pass membrane protein biogenesis.

Access to experimental X-ray diffraction image data is fundamental for validation and reproduction of macromolecular models and indispensable for development of structural biology processing methods. Here, we established a diffraction data publication and dissemination system, Structural Biology Data Grid (SBDG; data.sbgrid.org), to preserve primary experimental data sets that support scientific publications. Data sets are accessible to researchers through a community driven data grid, which facilitates global data access. Our analysis of a pilot collection of crystallographic data sets demonstrates that the information archived by SBDG is sufficient to reprocess data to statistics that meet or exceed the quality of the original published structures. SBDG has extended its services to the entire community and is used to develop support for other types of biomedical data sets. It is anticipated that access to the experimental data sets will enhance the paradigm shift in the community towards a much more dynamic body of continuously improving data analysis.

Tail-anchored (TA) membrane proteins destined for the endoplasmic reticulum are chaperoned by cytosolic targeting factors that deliver them to a membrane receptor for insertion. Although a basic framework for TA protein recognition is now emerging, the decisive targeting and membrane insertion steps are not understood. Here we reconstitute the TA protein insertion cycle with purified components, present crystal structures of key complexes between these components and perform mutational analyses based on the structures. We show that a committed targeting complex, formed by a TA protein bound to the chaperone ATPase Get3, is initially recruited to the membrane through an interaction with Get2. Once the targeting complex has been recruited, Get1 interacts with Get3 to drive TA protein release in an ATPase-dependent reaction. After releasing its TA protein cargo, the now-vacant Get3 recycles back to the cytosol concomitant with ATP binding. This work provides a detailed structural and mechanistic framework for the minimal TA protein insertion cycle., Approximately 5% of eukaryotic membrane proteins are anchored to the lipid bilayer by a single carboxy-terminal transmembrane domain (1-4) (TMD). These 'tail-anchored' proteins are found in virtually all cellular membranes [...]

Bat3: a chaperone to TA proteins Tail-anchored (TA) proteins have a single transmembrane domain at their C-termini and are post-translationally targeted to the endoplasmic reticulum via the cytosolic ATPase, TRC40. In this study, Mariappan et al. identify a conserved protein complex called Bat3 complex that is recruited to ribosomes and interacts with the transmembrane domain of newly released TA proteins, transferring them to TRC40 for subsequent targeting to the endoplasmic reticulum. Thus the Bat3 complex functions as a transmembrane-domain-specific chaperone that channels TA proteins to the TRC40 insertion pathway. Tail-anchored proteins have a single transmembrane domain at their carboxy termini and are post-translationally targeted to the endoplasmic reticulum via the cytosolic ATPase TRC40. These authors identify a conserved protein complex called Bat3 complex that is recruited to ribosomes, interacts with the transmembrane domain of newly released tail-anchored proteins and transfers them to TRC40 for subsequent targeting to the endoplasmic reticulum. Hundreds of proteins are inserted post-translationally into the endoplasmic reticulum (ER) membrane by a single carboxy-terminal transmembrane domain (TMD).sup.1. During targeting through the cytosol, the hydrophobic TMD of these tail-anchored (TA) proteins requires constant chaperoning to prevent aggregation or inappropriate interactions. A central component of this targeting system is TRC40, a conserved cytosolic factor that recognizes the TMD of TA proteins and delivers them to the ER for insertion.sup.2,3,4. The mechanism that permits TRC40 to find and capture its TA protein cargos effectively in a highly crowded cytosol is unknown. Here we identify a conserved three-protein complex composed of Bat3, TRC35 and Ubl4A that facilitates TA protein capture by TRC40. This Bat3 complex is recruited to ribosomes synthesizing membrane proteins, interacts with the TMDs of newly released TA proteins, and transfers them to TRC40 for targeting. Depletion of the Bat3 complex allows non-TRC40 factors to compete for TA proteins, explaining their mislocalization in the analogous yeast deletion strains.sup.5,6,7. Thus, the Bat3 complex acts as a TMD-selective chaperone that effectively channels TA proteins to the TRC40 insertion pathway., Author(s): Malaiyalam Mariappan [sup.1] , Xingzhe Li [sup.1] [sup.2] , Sandra Stefanovic [sup.1] , Ajay Sharma [sup.1] , Agnieszka Mateja [sup.3] , Robert J. Keenan [sup.3] , Ramanujan S. Hegde [...]

Targeting of newly synthesized membrane proteins to the endoplasmic reticulum is an essential cellular process. Most membrane proteins are recognized and targeted co-translationally by the signal recognition particle. However, nearly 5% of membrane proteins are 'tail-anchored' by a single carboxy-terminal transmembrane domain that cannot access the co-translational pathway. Instead, tail-anchored proteins are targeted post-translationally by a conserved ATPase termed Get3. The mechanistic basis for tail-anchored protein recognition or targeting by Get3 is not known. Here we present crystal structures of yeast Get3 in 'open' (nucleotide-free) and 'closed' (ADP x [A1F.sub.4.sup.-]-bound) dimer states. In the closed state, the dimer interface of Get3 contains an enormous hydrophobic groove implicated by mutational analyses in tail-anchored protein binding. In the open state, Get3 undergoes a striking rearrangement that disrupts the groove and shields its hydrophobic surfaces. These data provide a molecular mechanism for nucleotide-regulated binding and release of tail-anchored proteins during their membrane targeting by Get3., Eukaryotic cells have evolved sophisticated machinery to ensure high fidelity targeting and insertion of membrane proteins into intracellular organelles. For targeting to the endoplasmic reticulum (ER), the best understood mechanism [...]

The signal recognition particle (SRP) is a ribonucleoprotein machine, which in association with other cellular complexes direct proteins to the cell membrane. Research findings describe the key components of this machinery namely, the ribosome, signal sequences, particle receptor, and the translocon in the eukaryotic and prokaryaotic systems, which mediate SRP function.

A study was conducted to characterize the crystal structure of the signal sequence binding subunit of the signal recognition particle from Thermus aquaticus. A recombinant construct was expressed in Escherichia coli from a pET3c derivative while heavy atom sites were determined in isomorphous difference Patterson maps. Results indicated that the structure supported a deep groove with a flexible loop and side chains of conserved hydrophobic residues.

The structure of the nucleotide-free form of the NG fragment of the protein subunit of the signal-recognition particle is described. A compact network of interactions which may stabilize the nucleotide-free protein is formed by the side chains of the empty active-site pocket. Structurally similar N and G domains are present in the SR-alpha subunit. It appears that the N domain senses or controls the nucleotide occupancy of the GTPase domain.

Lung-reduction surgery may improve exercise capability and breathing difficulties in emphysema patients by increasing the elastic recoil of the lungs. Lung reduction involves the surgical removal of the most damaged lung tissue and may avoid continued hyperinflation of the lungs. Researchers evaluated the short-term impact of surgery in 20 patients with diffuse emphysema in terms of walking for six minutes and in terms of breathing problems. Nineteen cases were related to tobacco use. Three months after surgery, elastic recoil efficiency increased in 19 patients, corresponding to the ability to walk a greater distance in six minutes. All but two patients had improved breathing scores as determined by interviews. The records of the remaining two patients indicated a subjective improvement of dyspnea as well. The surgical impact on pulmonary mechanics should be addressed with respect to short-term benefits.