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Protein Transport in Cells
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 are complicated structures, containing a number of 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 and achieve their different structures? The answer to these questions is important for an understanding of how biological structures are generated and how they are propagated during cell division. The problem has relevance for the identification of the basis of diseases in which proteins are misdirected, misfolded, or degraded.
Proteins are directed to their different destinations by signals within their amino acid sequence. For example, proteins destined for export from the cell (secretory proteins) have hydrophobic signal sequences close to their amino termini. The signal sequence directs the polypeptide to the endoplasmic reticulum (ER) membrane, where it is transferred across the 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 system in bacteria and archaebacteria, where signal sequences similar to those in eukaryotes direct proteins across the cytoplasmic membrane. In recent years it has become clear that 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. We are interested in understanding the molecular mechanism of this retrotranslocation process.
Protein transport across the ER membrane can occur either co- or post-translationally. In the cotranslational pathway, transport occurs while the polypeptide chain is synthesized on a membrane-bound ribosome; in the post-translational pathway, the polypeptide chain is completed before being transferred across the ER membrane. Related pathways exist in bacteria and archaebacteria. In the past, we have used photocrosslinking to identify components that interact with the signal sequence of a nascent polypeptide chain and that are in proximity to the chain when it is transferred through the membrane. Central elements of the translocation site were identified: the mammalian translocating chain–associating membrane (TRAM) protein and the Sec61p complex, an evolutionarily conserved heterotrimeric membrane protein complex. With the purified components, we established a reconstituted system that reproduces the cotranslational mode of transport.
These early studies indicated that the Sec61p complex forms a protein-conducting channel. In addition, it binds the ribosome during cotranslational translocation and interacts specifically with signal sequences. Electron cryomicroscopy (performed in collaboration with Christopher Akey, Boston University) revealed that the channel sits underneath the ribosome, with a gap in the ribosome-channel junction likely providing a path for polypeptide segments into the cytosol.
Most membrane proteins are cotranslationally inserted into the ER membrane, but the precise mechanism is unclear. 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.
We have also reproduced post-translational translocation across the ER membrane with a defined reconstituted system consisting of a seven-component membrane protein complex, the Sec complex, and luminal BiP. The Sec complex comprises the Sec61p complex and a tetrameric Sec62/63p complex. We have shown that the signal sequence of a translocation substrate is specifically bound between TM segments 2 and 7 of Sec61p. The substrate is moved through the channel in a process in which BiP acts as an ATP-dependent molecular ratchet.
We have also conducted studies on the mechanism of protein translocation in bacteria. We have shown that SecA, the ATPase that pushes polypeptides through the channel, dissociates from a dimeric into a monomeric state during initiation of translocation. We have obtained the x-ray structure of monomeric SecA, which shows a large conformational change. More recently, we have shown that SecA-mediated translocation is mediated by oligomers of the SecY complex, but that a single SecY copy forms the channel. SecA binds through one of its domains to a nontranslocating SecY copy and moves the polypeptide chain through a neighboring SecY copy.
We determined the x-ray structure of an archaebacterial homolog of the Sec61p complex at 3.2-Å resolution (collaboration with the group of Stephen Harrison [HHMI, Harvard Medical School]). The structure shows the protein-conducting channel in its closed state, but it suggests mechanisms by which it is opened. 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 extracellular funnel of the hourglass is plugged by a short helix. During protein translocation, a signal sequence inserts itself into the mouth of the clamshell 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 how TM segments of membrane proteins move into the lipid phase and how the ribosome and SecA bind to the channel. Several of the predictions of the crystal structure have now been confirmed experimentally.
A major project in our lab concerns the mechanism of ERAD (ER-associated protein degradation). Proteins that are transported into the ER and cannot reach their native folded state are generally 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, travels backward along the secretory pathway, and is disassembled and unfolded when it arrives in the lumen of the ER. A fragment of the toxin, the A1 chain, is then retrotranslocated into the cytosol and ultimately opens channels in the plasma membrane, leading to massive secretion of chloride and water and consequent diarrhea. Our experiments have shown that cholera toxin is disassembled and unfolded by the ER enzyme protein disulfide isomerase (PDI). PDI acts as a novel chaperone that is regulated by a redox cycle and not by an ATPase cycle as used by conventional chaperones. PDI binds in its reduced state to the toxin, the complex binds to the ER membrane, and the toxin is then released from PDI upon oxidation by the enzyme Ero1.
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, ER membrane, or cytosolic side of the ER membrane. All three pathways converge at the Cdc48 ATPase complex.
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 strong defects of ER morphology. 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 are also interested in how DNA is moved during sporulation in Bacillus subtilis. Our recent results show that the ATPase SpoIIIE moves the DNA after cytokinesis through the two lipid bilayers. We also study how mRNA is transported in mammalian cells, and we have recently shown that the signal sequence–coding region directs the mRNA of secretory proteins to a novel nuclear export pathway.
A grant from the National Institutes of Health provided support for some of these projects.
Last updated: February 21, 2008
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