Research Summary
Tom Rapoport is interested in the mechanism by which proteins are transported across membranes and how organelles form and maintain their characteristic shapes.
Biological cells of higher organisms contain organelles that are separated from the cytosol by membranes. Each organelle has a characteristic shape and performs special functions that are carried out by different proteins. How do organelles receive specific sets of proteins, how do they clear themselves of misfolded proteins, and how do they achieve their different structures? The answers to these questions have relevance for diseases in which organelles are incorrectly shaped or proteins are misdirected, misfolded, or degraded.
Proteins are directed to their different destinations by signals within their amino acid sequence. Proteins destined for export from the cell (secretory proteins) have hydrophobic signal sequences, which trigger translocation of the polypeptide across the endoplasmic reticulum (ER) membrane. Many membrane proteins initially take the same route, but they are inserted into the lipid bilayer rather than being transported all the way across the membrane. Further signals in the polypeptide chain determine whether the protein stays in the ER or is transported to other destinations.
We are interested in the decisive first step during which proteins are translocated across or inserted into the ER membrane. Specifically, we would like to understand how signal sequences are recognized, how proteins are able to cross the membrane barrier, and how membrane proteins are integrated into the membrane and achieve their different topologies. These questions also apply to the translocation systems in bacteria and archaebacteria, where signal sequences similar to those in eukaryotes direct proteins across the cytoplasmic membrane.
Proteins transported into the ER that cannot reach their native folded states are transported back into the cytosol, where they are degraded by the proteasome. This process is called ERAD (for ER-associated protein degradation), and we are interested in understanding its molecular mechanism.
Several years ago we initiated a new field that addresses another major question in cell biology: how is the characteristic shape of an organelle generated? Specifically, we are studying the mechanisms by which the ER structure is generated and maintained.
Our early work on protein translocation across the ER membrane employed photo-crosslinking methods to identify proteins in proximity of translocating polypeptide chains. We found the component that forms the protein-conducting channel, an evolutionarily conserved membrane protein complex that is called the Sec61 complex in eukaryotes and the SecY complex in bacteria and archaea. Additional work led to the concept that the channel itself is just a passive pore; it needs to associate with partners that provide a driving force. In cotranslational translocation, the channel binds to the ribosome; the elongating polypeptide chain moves directly from the ribosome tunnel into the Sec61/SecY channel, as indicated by electron cryomicroscopy experiments (performed in collaboration with Christopher Akey, Boston University). In post-translational translocation in eukaryotes, the channel partners with another membrane protein complex, the Sec62/63 complex, as well as with the ER luminal protein BiP, a member of the Hsp70 family of ATPases. BiP acts as a molecular ratcheting molecule; it binds to the polypeptide chain on the luminal side of the channel and prevents it from moving back into the cytosol. Finally, in post-translational translocation in bacteria, the cytosolic SecA ATPase pushes polypeptides through the SecY channel. All modes of translocation can be reproduced with reconstituted systems composed of purified components.
Most membrane proteins are cotranslationally inserted into the ER membrane. We have shown that as soon as a transmembrane (TM) segment emerges from the ribosome, it exits the Sec61p channel laterally into the lipid phase. Our results show that the Sec61p channel mediates the partitioning of a TM segment into the lipid phase.
A major step in the field came from the determination of the first x-ray structure of a protein-conducting channel (collaboration with Stephen Harrison [HHMI, Harvard Medical School]). Viewed from the cytosol, the channel looks like a clamshell, and in a cutaway view from the side, it looks like an hourglass with a constriction in the middle of the membrane. The cytosolic funnel of the hourglass is empty, whereas the extracellular funnel is plugged by a short helix. During protein translocation, a signal sequence inserts itself into the mouth of the clamshell, the lateral gate, and destabilizes interactions that keep the plug in the center of the molecule. The plug moves out of the way, and the polypeptide chain then passes through the constriction from the cytosolic to the extracellular funnel. The structure also suggests that TM segments of membrane proteins move through the lateral gate into the lipid phase. Several of the predictions of the crystal structure have now been confirmed experimentally.
In recent years, we have concentrated on the mechanism of SecA-mediated protein translocation in bacteria. We have shown that SecA dissociates from a dimeric into a monomeric state during initiation of translocation. Disulfide bridge crosslinking experiments demonstrated that a single copy of the SecY complex forms the channel, but translocation is mediated by SecY oligomers. SecA binds through one of its domains to a nontranslocating SecY copy and moves the polypeptide chain through a neighboring SecY copy.
Our x-ray structures of SecA bound to the SecY complex suggest how the ATPase moves polypeptides through the channel. SecA has a two-helix finger that is inserted into the cytoplasmic opening of the SecY channel. The two-helix finger of SecA could move a polypeptide chain into the channel, with a tyrosine at the fingertip providing the major contact with the substrate, a mechanism that is analogous to that suggested for hexameric protein-translocating ATPases. Coordinated with the movements of the two-helix finger, a "clamp" in SecA that is formed by the rotation of a domain would tighten and widen above the SecY pore. Several of these predictions have now been confirmed experimentally.
A major project in our lab concerns the mechanism of ERAD, a process in which misfolded ER proteins are transported back into the cytosol, polyubiquitinated, and degraded by the proteasome. The cellular pathway is hijacked by certain viruses and toxins. For example, the human cytomegalovirus protein US11 triggers the degradation of the antigen-presenting major histocompatibility complex class I molecules. We have used a permeabilized cell system to study this process. Our results show that the substrate is polyubiquitinated and moved into the cytosol by a member of the AAA family of ATPases, p97 (called Cdc48p in yeast), that functions together with two cofactors. In subsequent experiments, we discovered a membrane receptor of the ATPase complex, consisting of Derlin-1, a homolog of yeast Der1p, which is involved in the retrotranslocation of misfolded ER luminal proteins, and VIMP. Other experiments showed that both the ATPase complex and the polyubiquitination machinery are recruited jointly to the retrotranslocation site in the ER membrane.
We have studied early stages of retrotranslocation with cholera toxin as a model. The toxin is taken up by intestinal cells and releases a fragment, the A1 chain, from the ER into the cytosol. Our experiments have shown that cholera toxin is disassembled and unfolded by the ER enzyme protein disulfide isomerase (PDI).
We have performed a comprehensive analysis of the ERAD pathways in yeast. This analysis identified distinct ubiquitin-ligase complexes that define different pathways for proteins with misfolded domains in the ER lumen (ERAD-L), ER membrane (ERAD-M), or cytosolic side of the ER membrane (ERAD-C). All three pathways converge at the Cdc48 ATPase complex. These experiments led to the identification of most, if not all, ERAD components.
Another major project in our lab addresses the mechanism by which the ER structure is generated and maintained. Using an in vitro system that recapitulates the formation of an ER network with Xenopus egg membranes, we identified reticulon 4a as a component required for ER tubule formation. The reticulons, a ubiquitous protein family, are localized exclusively to the tubular ER. DP1/Yop1, a conserved interaction partner of the reticulons, also localizes exclusively to the tubular ER, and the absence of these proteins in yeast cells leads to the loss of tubular ER. Purified yeast reticulon or Yop1p deforms reconstituted proteoliposomes into tubules, indicating that these proteins are not only necessary, but also sufficient for shaping membranes into tubules. We have proposed that the reticulons and DP1/Yop1 belong to a novel class of membrane proteins that localize to and stabilize highly curved membrane structures.
We have identified a class of dynamin-like GTPases, atlastins and Sey1p, which seem to be involved in ER network formation. These membrane-bound GTPases interact with the tubule-shaping proteins and localize to the tubular ER. Depletion and overexpression experiments in mammalian cells, and antibody inhibition experiments in the Xenopus in vitro system, are consistent with a role of the atlastins in homotypic ER membrane fusion. The yeast protein Sey1p and its plant homologs may have a similar function. Mutations in atlastin-1 cause hereditary spastic paraplegia, a disease characterized by the progressive shortening of the long axons of motor neurons, possibly by compromising ER morphology.
Over the years, we have also studied the mechanism by which BiP binds its peptide substrates, the regulation of the molecular motor kinesin, the differentiation between rough and smooth ER in Caenorhabditis elegans, the mechanism by which nonidentical compartments are generated in bi-directional vesicular transport systems, the mechanism by which DNA is transported across membranes during sporulation in Bacillus subtilis, the mechanism by which mRNA is exported from the nucleus, and, most recently, the structure and function of vitamin K epoxide reductase (VKOR), an enzyme that plays an important role in blood coagulation.
A grant from the National Institutes of Health provided support for some of these projects.
As of March 05, 2010
