Tom A. Rapoport, Ph.D. - US grants

The long-term goal of this project is to understand the molecular mechanism by which proteins are transported across or are inserted into the mammalian endoplasmic reticulum (ER) membrane. We have established reconstituted proteoliposome and soluble systems with purified membrane protein components which reproduce the overall translocation process and partial reactions. The minimum translocation apparatus of the ER membrane comprises only the heterotrimeric Sec61p complex, the TRAM protein, and the SRP receptor. We now propose to perform a thorough analysis of the mechanism of translocation. Specifically, we will use questions: 1. How is cotranslational translocation initiated? 2. How do ribosomes interact with the Sec61p channel? 3. How are membrane proteins inserted into the lipid bilayer? 4. What is the function of the mammalian homologs of Sec62p and Sec63p? These studies will contribute significantly to our understanding of the first and decisive step in the biosynthesis of a large of class proteins, proteins of the plasma membrane, of lysosomes, and of all organelles of the secretory pathway.

The application seeks to understand the mechanism of posttranslational translocation across the membrane of the endoplasmic reticulum of Saccharomyces cerevisiae. Specific Aim 1 addresses the mechanism of targeting of substrates. The presence and importance of cytosolic chaperones or targeting molecules will be assessed. The second Specific Aim will determine the role of the signal sequence on the gating of the translocating channel. It will test the hypothesis that the signal sequence displaces a component of the translocon, and as a result, opens the translocation channel.. Specific Aim 3 is to determine the stoicheometry of the components of the translocon. Estimations of 3 or 4 Sec61p complexes comprising the translocation channel have been made based on electron micrographic techniques, and this will be tested biochemically. A role of Sec62p in catalyzing assembly will be tested. Finally, Specific Aim 4 seeks to understand the role of luminal Hsp70 in the translocation process. It will test the hypothesis that this protein works as a molecular ratchet rather than a machine that "pulls" the protein through the membrane.

The application seeks to understand the mechanism of posttranslational translocation across the membrane of the endoplasmic reticulum of Saccharomyces cerevisiae. Specific Aim 1 addresses the mechanism of targeting of substrates. The presence and importance of cytosolic chaperones or targeting molecules will be assessed. The second Specific Aim will determine the role of the signal sequence on the gating of the translocating channel. It will test the hypothesis that the signal sequence displaces a component of the translocon, and as a result, opens the translocation channel.. Specific Aim 3 is to determine the stoicheometry of the components of the translocon. Estimations of 3 or 4 Sec61p complexes comprising the translocation channel have been made based on electron micrographic techniques, and this will be tested biochemically. A role of Sec62p in catalyzing assembly will be tested. Finally, Specific Aim 4 seeks to understand the role of luminal Hsp70 in the translocation process. It will test the hypothesis that this protein works as a molecular ratchet rather than a machine that "pulls" the protein through the membrane.

DESCRIPTION (provided by applicant): The goal of this project is to understand in mechanistic terms how proteins are transported across membranes. Previous work has demonstrated that translocation occurs through a protein-conducting channel that is formed from a heterotrimeric membrane protein complex (called Sec61p complex in eukaryotes, SecYEG complex in bacteria, and SecYEbeta complex in archaebacteria). Depending on the interacting partner, the channel can function in four different translocation pathways: 1. Co-translational translocation, 2. Post-translational translocation in eukaryotes, 3. Post-translational translocation in bacteria, and 4. Retrotranslocation from the endoplasmic reticulum (ER) into the cytosol. This proposal addresses key aspects of all four translocation modes, with particular emphasis on the least understood retro-translocation pathway. Specifically, we will address the following questions: 1. How is the protein-conducting channel assembled and gated? The planned experiments will clarify how the ribosome binds to the channel in co-translational translocation, how the channel is assembled from several copies of the heterotrimeric membrane protein complex, and how it is opened for translocation. We will also test whether the ER membrane is permeable for small molecules. 2. How does SecA move polypeptides through the channel? We will address the mechanism of posttranslational translocation in bacteria. Specifically, we will test the hypothesis that SecA functions analogously to monomerie helicases to push polypeptides through the channel. 3. How is retro-translocation initiated? We will investigate whether the unfolding of cholera toxin is coupled to its subsequent retro-translocation through the ER membrane. This will involve experiments that address the role of protein disulfide isomerase (PDI) and its oxidase Erol in retro-translocation. 4. How are proteins moved from the ER membrane into the cytosol? The goal of these experiments is to understand how the HMC class I heavy chain is moved into the cytosol during retro-translocation induced by the human cytomegalovirus protein US 11. The planned experiments will identify the components required for poly-ubiquitination of the heavy chain and will test the role of the AAA ATPase p97 and of its partner proteins in extracting proteins from the ER membrane.

DESCRIPTION (provided by applicant): The goal of this project is to understand in mechanistic terms how proteins are transported across membranes. Both secretory and membrane proteins are translocated from the cytosol across the membrane through a channel that is formed from a heterotrimeric membrane protein complex, the Sec61p complex in eukaryotes and the SecY complex in bacteria and archae. We have determined X-ray structures of the SecY complex alone and when associated with the ATPase SecA, which have led to new insights and provide the basis for part of the present proposal. In eukaryotes, there is a translocation pathway in the reverse direction, called ERAD (for ER associated degradation), which is used to degrade misfolded ER proteins. We have identified most, if not all, components involved in ERAD, paving the way for mechanistic studies. Here, we will address key aspects of translocation with specific emphasis on the following questions: 1. How are proteins cotranslationally translocated and how is the membrane barrier for small molecules maintained during the process? Based on a new method to generate cotranslational translocation intermediates in intact E. coli cells and the ability to purify ribosome/nascent chain/channel complexes, we will determine how many copies of SecY are required for translocation and will use electron microscopy to elucidate how the active channel binds to the ribosome. We will investigate how the channel maintains the membrane barrier for small molecules during translocation. 2. What is the mechanism of posttranslational translocation in bacteria? We will clarify the mechanism by which SecA moves polypeptides through the channel. We will address the unexplored role of the SecDFYajC complex and test its involvement in mediating the effect of a membrane potential on translocation. 3. What is the molecular mechanism of ERAD? We will probe the path of a luminal ERAD (ERAD-L) substrate and determine how it is recognized. Based on preliminary results that indicate a crucial role for the ubiquitin ligase Hrd1p, we will purify the protein, and reconstitute it together with its partner proteins. We will develop a purified component system that recapitulates subreactions or even the entire ERAD-L process. PUBLIC HEALTH RELEVANCE: The mechanism of protein translocation is of great medical importance. Drugs that inhibit signal sequence binding can be used for therapeutic intervention in chronic inflammatory diseases. A large number of diseases, including cystic fibrosis and a1-antitrypsin deficiency, are caused by mutations that result in the misfolding of ER proteins and their subsequent degradation in the cytosol. The pathway is also hijacked by certain viruses and toxins, and a better understanding may lead to new drugs allowing interference.

PROJECT SUMMARY The goal of this project is to define the molecular mechanism by which lung surfactant protein B (SP-B) functions in respiration, and to develop an optimized surfactant mixture that could ultimately be used as a therapeutic for the treatment of Acute Respiratory Distress Syndrome (ARDS). SP-B is the only surfactant protein essential for breathing. It is made in alveolar type II cells as a precursor containing three related domains and is then proteolytically processed into the individual domains (SP-BN, SP-BM, SP-BC), with the middle domain (SP-BM) currently considered to be the ?mature? protein. Surfactant originates from the secretion of lamellar bodies (LB), in which membrane sheets are densely stacked. The exported lipid bilayers then form a lipid monolayer at the air-water interface, which reduces surface tension and facilitates breathing. The exact functions of SP-BM and the other SP-B domains are unknown. Based on crystal structures, biochemical and cell biology experiments, we discovered that SP-BN, which had been largely ignored, is a non-specific lipid transfer protein in lungs. We also found that reconstitution of purified SP-BM into liposomes results in structures that have a striking resemblance to human LBs, suggesting that the entire organelle can be generated from a single protein. Further preliminary results indicate that intratracheal administration of purified SP-BN together with liposomes has a beneficial effect in a mouse model of ARDS. Based on these preliminary results, we will address the following questions: Specific aim #1: What are the functions of SP-BN and SP-BC? We will use purified SP-BN to elucidate the mechanism of lipid transfer and test whether purified SP-BC augments the activity of SP-BN. CRISPR and infection with adeno-associated virus (AAV) constructs will be used to test in mice whether SP-BN has a role in respiration and LB formation. We will use bronchoscopy of ARDS patients and control individuals to test for the presence of SP-BN in lavage fluid. Specific aim #2: What is the function of SP-BM? We will address the mechanism by which SP-BM forms LB-like structures using 2D crystallization and biochemical techniques. We will establish an expression and purification system for SP-BM, which has been a major bottleneck in the field, and determine structures of SP-BM by X-ray crystallography or cryo-EM. We will test whether SP-BN and SP-BC act synergistically with SP-BM to form LB-like structures in vitro. Specific aim #3: Can we generate a surfactant with therapeutic value? We will test whether SP-BN, SP-BM, and possibly SP-BC, together constitute the active extracellular surfactant. Intratracheal administration of a mixture of purified proteins will test whether mice expressing SP- B under an inducible promoter retain normal lung function in the absence of inducer in the diet. The mixture will also be tested in models of ARDS, in which lung injury is induced by either lipopolysaccharide or acid.