We study the mechanisms regulating the synthesis, translocation, and maturation of secretory and membrane proteins at the mammalian endoplasmic reticulum (ER). A complex macromolecular assembly at the ER, termed the translocon, serves as a protein-conducting channel where substrates enter the secretory pathway. The translocon participates in diverse cellular activities that range from the import of secretory proteins to the topogenesis and assembly of complex multi-spanning membrane proteins to the export of misfolded substrates from the ER to the cytosol for degradation. Still largely unknown are the mechanisms that allow the shared translocon to accommodate such an extensive range of substrates efficiently yet accurately. The principal goal of our ongoing studies is to define the molecular mechanisms and components of the translocon that recognize the information in the primary sequence of the translocon’s substrates and thus mediate their proper vectorial transport, asymmetric topogenesis, and membrane integration. By comparing the steps involved in the biosynthesis of normal versus disease-associated variants of secretory and membrane proteins, we are formulating and testing in vivo a set of hypotheses about the molecular basis of particular diseases of the early secretory pathway.
Prion protein cell biology and neurodegeneration
Rane, Chakrabarti, Ashok, Hegde N, Hegde R; in collaboration with Feigenbaum
The prion protein (PrP), a brain glycoprotein involved in various neurodegenerative diseases, has proven to be a particularly instructive example of complex and highly regulated translocation. In addition to PrP’s notoriety as the putative “protein-only” infectious agent in prion diseases, its biogenesis at the ER is unusual in that an initially homogeneous cohort of nascent PrP chains gives rise to four distinct topologic forms: a fully translocated form (termed secPrP), two transmembrane forms that span the membrane in opposite orientations (NtmPrP and CtmPrP), and a cytosolic form (cyPrP). In vivo studies have revealed that even a slight overrepresentation of the CtmPrP topologic form results in the development of neurodegenerative disease in both mouse model systems and naturally occurring human disease. Furthermore, cyPrP can be both aggregation-prone and neurotoxic under some circumstances.
To gain insight into how the variants are initially generated, we are dissecting the pathways of PrP biogenesis and degradation. Our results indicate that the decisive event in avoiding the generation of both potentially harmful forms of PrP (CtmPrP and cyPrP) is the signal sequence–mediated translocation of the N-terminus of PrP into the ER lumen. The use of a constitutive, highly efficient signal sequence can substantially reduce the generation of CtmPrP and cyPrP. The consequences of this manipulation in a cultured neuronal cell line are a marked reduction of cytotoxic and aggregation-prone variants of PrP and protection from apoptotic cell death. The results define a pathway for the normal biogenesis of PrP and demonstrate that the total cellular burden of cytotoxic forms of PrP is controlled primarily during PrP’s initial translocation into the ER. We have created transgenic mice to determine whether CtmPrP-mediated and cyPrP-mediated neurodegeneration can be averted in vivo by modulating this newly discovered step during PrP biogenesis. We are also investigating the pathways by which the cell normally metabolizes various forms of PrP in order to determine whether modulation of these metabolic events is involved in the progression of neurodegeneration. We expect that a combination of defects in biosynthesis and/or clearance of certain forms of PrP collaborates to cause eventual neuronal dysfunction and death. Conversely, manipulation of these events may be able to slow or reverse the neurodegenerative process.
Parallel biochemical studies employing the solubilization, fractionation, and reconstitution of ER membrane proteins have demonstrated that regulatory trans-acting factors are absolutely required for PrP to be synthesized in the proper ratio of its topologic forms. We have now purified two of these factors and identified them as the translocon-associated protein complex (TRAP) and protein disulfide isomerase (PDI). Analysis of PrP translocation intermediates suggests that TRAP and PDI act sequentially to facilitate translocation of PrP’s N-terminus into the ER lumen, the decisive event in determining PrP’s topology. Ongoing studies are investigating the role of the newly discovered factors in the biogenesis of other substrates and their potential role in the pathogenesis of PrP-associated neurodegeneration.
Hegde RS, Rane NS. The molecular basis of prion protein-mediated neuronal damage. In: Brown DR, ed. Neurodegeneration and Prion Disease. New York: Springer, 2005:407-450.
Rane NS, Yonkovich JL, Hegde RS. Protection from cytosolic prion protein toxicity by modulation of protein translocation. EMBO J 2004;23:4550-4559.
Tremblay P, Ball HL, Kaneko K, Groth D, Hegde RS, Cohen FE, DeArmond SJ, Prusiner SB, Safar JG. Mutant PrPSc conformers induced by a synthetic peptide and various prion strains. J Virol 2004;78:2088-2099.
Regulation of protein biogenesis and function by signal sequences
Kang, Sharma, Levine,2 Eagan; in collaboration with Sharan, Tessarollo
Our discovery that the N-terminal signal sequence of PrP regulates PrP’s topology established a new function for this domain independent of its well-studied role in protein targeting. We subsequently developed and used a novel assay for signal-mediated translocon gating to demonstrate that signal sequences display a remarkable degree of variation in initiating nascent chain access to the lumenal environment. We found that substrate-specific properties of signals were evolutionarily conserved, functionally matched to their respective mature domains, and important for the proper biogenesis of some proteins. A recent analysis of several naturally occurring disease-associated mutants in signal sequences has revealed that many of the mutants are altered in their gating function and are not involved in targeting as previously assumed. Thus, we discovered that the long-observed sequence variations of signals do not simply represent functional degeneracy but instead also encode differences in translocon gating that are critical to the proper biogenesis of the attached substrate. We are currently investigating whether the cell exploits substrate-specific properties of signal sequences to regulate the subcellular localization of certain proteins, such as calreticulin, that are known to be present in several compartments. Furthermore, we are identifying conditions, such as ER stress, during which the cell adaptively modulates the translocation of some but not other substrates. Using RNAi-based screens in cell culture, the mechanism of such regulation and its associated factors are under investigation.
Hegde RS. Protein translocation across the endoplasmic reticulum. In: Eichler J, ed. Protein Movement Across Membranes. Georgetown, TX: Landes Bioscience, 2005:1-18.
Levine CG, Mitra D, Sharma A, Smith CL, Hegde RS. The efficiency of protein compartmentalization into the secretory pathway. Mol Biol Cell 2005;16:279-291.
Shaffer KL, Sharma A, Snapp EL, Hegde RS. Regulation of protein compartmentalization expands the diversity of protein function. Dev Cell2005;9:545-554.
Structural and functional analysis of the TRAP complex
Hegde R; in collaboration with Akey, Snapp
The search for factors involved in PrP topogenesis led to our purification of the TRAP complex, a set of four proteins with a previously unknown function. Our recent studies established that TRAP is required for the translocation of some but not other substrates. We discovered that substrate specificity is encoded in the signal sequence. Remarkably, TRAP specifically aids vectorial transport of substrates whose signal sequences, after mediating ER targeting, are delayed in their gating of the translocon. Thus, it appears that TRAP is a key component of the translocation machinery that aids in decoding substrate-specific information in signal sequences. Using two approaches, our current studies of TRAP function focus on understanding the molecular mechanism by which TRAP facilitates signal recognition, substrate translocation, and protein topogenesis. In collaboration with the laboratory of Christopher Akey, we are using cryo-electron microscopy to compare the structures of ribosome-translocon complexes that have been prepared both with and without the TRAP complex. The studies are not only providing the initial structural views of the TRAP complex but are also defining its relative position within the translocon. In parallel, we are investigating the consequence in vivo of RNAi-mediated suppression of TRAP expression in cultured cells. We expect that the structural information combined with the functional analyses will provide insight into the mechanisms by which TRAP can regulate protein translocation in a substrate-specific manner.
Menetret JF, Hegde RS, Heinrich SU, Chandramouli P, Ludtke SJ, Rapoport TA, Akey CW. Architecture of the ribosome-channel complex derived from native membranes. J Mol Biol 2005;348:445-457.
Small-molecule inhibitors of protein translocation
Rane, Hegde R; in collaboration with Taunton
To develop new methods and probes for protein translocation, we have been developing pharmacologic methods of modulating the translocation process in vivo. We recently synthesized and characterized a novel small-molecule inhibitor of co-translational protein translocation and demonstrated that, both in vitro and in cultured cells, it inhibits the translocation of some but not other proteins. Remarkably, the substrate specificity was found to be encoded in the signal sequence. Thus, simply the choice of signal can confer to any protein of interest the sensitivity or resistance to the inhibitor. We have identified the target of the inhibitor; it appears to be the Sec61 complex, the central component of the protein translocation channel. Our methods and findings now open the way to the selective and potent modulation of the translocation of individual substrates in live cells. We are applying such an approach to studying the role of protein translocation in various cellular events such a protein aggregation and toxicity, protein degradation, and the cellular response to ER stress.
Garrison JL, Kunkel EJ, Hegde RS, Taunton J. A substrate-specific inhibitor of protein translocation into the endoplasmic reticulum. Nature 2005;436:285-289.
Visualization of ER and translocon organization in cells
Hegde R; in collaboration with Lippincott-Schwartz, Snapp
A qualitatively different facet of protein biogenesis is the location within the ER of various associated events. As noted above, the translocon participates in diverse cellular activities. While the current approaches to understanding protein translocation use biochemical systems that largely result in the loss of spatial relationships, little is known about whether all translocon-associated activities are homogeneously distributed throughout the ER or whether are they organized and regulated in the spatial and temporal dimensions to meet the cell’s changing needs. In collaboration with the laboratory of Jennifer Lippincott-Schwartz, we are using biophysical techniques such as fluorescence resonance energy transfer (FRET) to probe in situ the molecular organization of the components of the translocation machinery. In initial studies, we used analysis of FRET between subunits of the Sec61p complex, a principal component of the ER protein translocon, to monitor directly the assembly state of the translocon in cells. Our studies revealed that while the translocon can be assembled from its components in response to ligands for protein translocation in biochemical systems, it does not disassemble and reassemble between successive rounds of transport in vivo. Instead, an actively engaged translocon is distinguished from a quiescent translocon by conformational changes that can be directly detected by differences in FRET. By correlating the formation of particular protein complexes with biochemical activities, we endeavor to visualize directly the functional segregation and organization of the ER and to monitor potential changes in it during cellular metabolism, development, or disease pathogenesis.
Snapp EL, Hegde RS, Francolini M, Lombardo F, Colombo S, Pedrazzini E, Borgese N, Lippincott-Schwartz J. Formation of stacked ER cisternae by low affinity protein interactions. J Cell Biol 2003;163:257-269.
Snapp EL, Reinhart GA, Bogert BA, Lippincott-Schwartz J, Hegde RS. The organization of engaged and quiescent translocons in the endoplasmic reticulum of mammalian cells. J Cell Biol 2004;164:997-1007.
Mechanism of tail-anchored protein insertion
Stefanovic; in collaboration with Borgese
A commonly used mechanism for subcellular localization of a membrane protein is the presence of a single C-terminal hydrophobic domain capable of insertion into the lipid bilayer. Such tail-anchored (TA) proteins are found on all intracellular membranes exposed to cytosol and are involved in a remarkably diverse range of physiologic processes, from intracellular trafficking to protein degradation to programmed cell death. Thus, deciphering the molecular details underlying the selective membrane insertion and trafficking of TA proteins is critical to understanding a wide range of cell-biological and physiological processes. Nonetheless, the mechanisms used by TA proteins to arrive at and insert into their target membrane are largely unresolved. We have been using a biochemical approach to identify factors that regulate the membrane insertion of TA proteins and have found that for one model protein, cytochrome b5, protein factors are not obligatorily involved. Instead, the lipid composition of the target membrane appears to influence insertion, with cholesterol as at least one factor. By contrast, other model TA proteins appear to require protein factors in both the cytosol and target membrane. We are now identifying the various factors by using fractionation and functional reconstitution approaches.
Brambillasca S, Yabal M, Soffientini P, Stefanovic S, Makarow M, Hegde RS, Borgese N. Transmembrane topogenesis of a tail-anchored protein is modulated by membrane lipid composition. EMBO J 2005;24:2533-2542.
Regulation of membrane protein integration
Alken, Hegde R
The basic steps and minimal machinery needed for the biosynthesis of simple secretory and membrane proteins have been largely elucidated. However, essentially nothing is known about how complex multispanning proteins are properly produced with high fidelity. This issue is of particular interest because complex multispanning proteins include some of the most important cell surface molecules, namely, ion channels, transporters, and receptors, which are defective in many diseases and therefore also are targets for many therapeutic drugs. At least three issues need to be resolved: determining whether the production of complex multispanning proteins can be functionally reconstituted in a biochemical system amenable to fractionation; identifying the functional requirements for the production of multispanning membrane proteins; and specifying the steps in the biosynthesis of proteins that are defective in disease-associated mutants. We are using several G protein–coupled receptors as model membrane proteins to begin addressing these issues. We have so far reconstituted the biosynthesis and insertion of at least some receptors in an in vitro system and are using the system’s manipulability to identify factors involved in key biosynthetic steps. We expect that our studies will provide critical insight into the mechanisms of membrane protein assembly and the consequences of dysregulation of these mechanisms for disease pathogenesis.
Karsten V, Hegde RS, Sinai AP, Yang M, Joiner K. Transmembrane domain modulates sorting of membrane proteins in Toxoplasma gondii. J Biol Chem 2004;279:26052-26057.
1Left NICHD August 2005.
2Corinna Levine, BS, former Predoctoral Fellow, left NICHD August 2004.
Collaborators
Christopher Akey, PhD, Boston University School of Medicine, Boston, MA
Nica Borgese, PhD, CNR, Milan, Italy
Lionel Feigenbaum, PhD, SAIC Frederick, Inc., NCI, Frederick, MD
Jennifer Lippincott-Schwartz, PhD, Cell Biology and Metabolism Branch, NICHD, Bethesda, MD
Shyam Sharan, PhD, Center for Cancer Research, NCI, Frederick, MD
Erik Snapp, PhD, Albert Einstein College of Medicine, New York, NY
Jack Taunton, PhD, University of California San Francisco, San Francisco, CA
Lino Tessarollo, PhD, Center for Cancer Research, NCI, Frederick, MD
For further information, contact hegder@mail.nih.gov.