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REGULATION OF SECRETORY AND MEMBRANE PROTEIN BIOGENESIS
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Ramanujan S. Hegde, MD, PhD, Head, Unit on Protein Biogenesis Ajay Sharma, PhD, Research Fellow |
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| 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 topogenesis and assembly of complex multispanning membrane proteins, to the export of misfolded substrates from the ER to the cytosol for degradation. The mechanisms that allow this mutually shared translocon to accommodate such an extensive range of substrates both efficiently and accurately remain largely unknown. 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 its substrates, thus mediating their proper vectorial transport, asymmetric topogenesis, and membrane integration. By delineating the steps during the biosynthesis of normal versus disease-associated variants of secretory and membrane proteins, we are formulating and testing hypotheses in vivo regarding the molecular basis of particular diseases of the early secretory pathway. Molecular mechanisms of prion protein topogenesis Kim, Fons, Hegde 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 its notoriety as the putative "protein-only" infectious agent in prion diseases, the biogenesis of PrP at the ER is unusual in that an initially homogeneous cohort of nascent PrP chains gives rise to three distinct topologic forms: a fully translocated form (termed secPrP) and two transmembrane forms that span the membrane in opposite orientations (NtmPrP and CtmPrP). 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. Our current efforts focus on dissecting the mechanisms that direct an initially homogeneous cohort of nascent PrP polypeptides into multiple topologic forms. We recently discovered that segregation of nascent PrP into different topologic forms is critically dependent on the precise timing of signal sequence-mediated initiation of N-terminus translocation. Consequently, this segregation step could be experimentally tuned to modify PrP topogenesis, including complete reversal of the elevated CtmPrP caused by disease-associated mutations in the transmembrane domain. We are initiating studies in transgenic mice to determine whether CtmPrP-mediated neurodegeneration can be averted by modulating this newly discovered step during PrP biogenesis. 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 its topology. Ongoing studies are investigating the role of these newly discovered factors in the biogenesis of other substrates and their potential role in the pathogenesis of PrP-associated neurodegeneration. Fons RD, Bogert BA, Hegde RS. Substrate-specific function of the translocon-associated protein complex during translocation across the ER membrane. J Cell Biol 2003;160:529-539. the translocation channel. Mol Biol Cell 2002;13:3775-3786. Modulation of prion protein cytotoxicity in vivo Rane, Hegde A principal strategy for controlling the progression of various neurodegenerative diseases is to reduce the total cellular burden of the potentially cytotoxic forms of the responsible protein. In diseases associated with the prion protein (PrP), neurodegeneration may be mediated by several variant isoforms that include a transmembrane form (termed CtmPrP; see above) and a more recently described cytoplasmic form. To gain insight into how these variants are initially generated, we have dissected the pathway of PrP biogenesis at the ER (see above). Our results indicate that the decisive event in avoiding the generation of both of these forms is the signal sequence-mediated translocation of the N-terminus of PrP into the ER lumen. This critical step in PrP translocation is modulated by TRAP, a heterotetrameric membrane protein of previously unknown function, and ER lumenal chaperones. By using a constitutive, highly efficient signal sequence that does not depend on these factors for efficient N-terminus translocation, the generation of CtmPrP and cytosolic PrP can be substantially reduced. 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. These 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 its initial translocation into the ER. Some of our ongoing studies focus on examining the consequences in transgenic mice of altering the cellular burden of cytoplasmic forms of PrP. In particular, we wish to ask whether reducing the amounts of this potentially neurotoxic species of PrP confers protection against neurodegeneration during the course of transmissible prion disease. Hegde RS, Rane NS. Prion protein trafficking and the development of neurodegeneration. Trends Neurol Sci 2003;26:337-339. Mechanisms of calreticulin multifunctionality Shaffer, Sharma, Hegde The generation of multiple topologic forms of the prion protein has raised the provocative possibility that a single protein can exist in multiple spatially and functionally discrete populations. We have therefore sought to identify and characterize examples of proteins whose translocation into the ER is productively regulated to generate multiple functional forms. To this end, we have been studying the protein calreticulin, an abundant calcium-binding chaperone of the ER lumen. Remarkably, this protein has also been implicated in functions outside the ER, such as nuclear export, binding to and modulating activity of steroid hormone receptors, and binding to and influencing integrin signaling. How this multifunctional protein might perform these functions in multiple cellular compartments remains unknown. We have recently discovered that the signal sequence of calreticulin is naturally designed in a manner that allows a small but discrete population of the protein to be retained in the cytoplasm. This cytoplasmic population can be reduced by using a signal sequence from another protein, prolactin. Thus, we have identified a potential mechanism by which a protein can be made in more than one population and a means to manipulate such heterogeneity. We are now examining the relative contributions of the different populations of calreticulin to each of the multiple functions associated with the protein. We wish to determine if and how calreticulin translocation is regulated by the cell to change its localization and, hence, function to meet changing cellular demands with stress, cell cycle, differentiation, or disease pathogenesis. Regulation of protein biogenesis by signal sequences Yonkovich, Mitra, Levine Our discovery that the N-terminal signal sequence of PrP regulates its topology established a new function for this domain independent of its well-studied role in protein targeting. Using signal sequence mutants that uncouple its targeting and post-targeting functions, we demonstrated that the post-targeting function lies in gating the translocation channel to provide the nascent polypeptide access to the ER lumen. We subsequently developed and used a novel assay for signal-mediated translocon gating to demonstrate that signal sequences display remarkable variability in initiating nascent chain access to the lumenal environment. We found that such substrate-specific properties of signals are 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 revealed that many of them are altered in their gating function and not involved in targeting as previously assumed. Thus, we have discovered that the long-observed sequence variations of signals do not simply represent functional degeneracy but instead encode differences in translocon gating that are critical to the translocon's attached substrate. We are currently investigating whether the substrate-specific properties of signal sequences have been exploited by the cell to regulate the subcellular localization of certain proteins, such as calreticulin, that have been shown to be present in multiple compartments. Hegde RS. Targeting and beyond: new roles for old signal sequences. Mol Cell 2002;10:697-698. channel in a substrate-specific manner. Dev Cell 2002;2:207-217. regulation: a new view of secretory and membrane protein folding. Bioessays 2002;24:741-748. Structural and functional analysis of the TRAP complex Fons, Schwartz; in collaboration with Akey 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 establish that TRAP is required for the translocation of some, but not all, substrates and that substrate specificity is encoded in the signal sequence. Remarkably, TRAP specifically aids vectorial transport of substrates whose signal sequences, after mediating targeting to the ER, are delayed in their gating of the translocon. Thus, it appears that TRAP is a critical component of the translocation machinery that aids in decoding the substrate-specific information in signal sequences. Our current studies of TRAP function are following two approaches to understand the molecular mechanism by which it facilitates signal recognition, substrate translocation, and protein topogenesis. In collaboration with Christopher Akey's group, we are using cryo-electron microscopy to compare the structures of ribosome-translocon complexes that have been prepared with and without the TRAP complex. The studies are providing the initial structural views of not only the TRAP complex but also of 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. It is expected 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. Visualization of translocon organization in cells Hegde; in collaboration with Lippincott-Schwartz, Snapp A qualitatively different facet of protein biogenesis is the question of where within the ER various events occur. The translocon participates in diverse cellular activities that range from the import of secretory proteins, to topogenesis and assembly of complex multispanning membrane proteins, to the export of misfolded substrates from the ER to the cytosol for degradation. We wish to determine whether all these translocon-associated activities are homogeneously distributed throughout the ER, or whether they are organized and regulated in spatial and temporal dimensions to meet the changing needs of the cell. Currently, there is little or no insight into this question largely because the current approaches to understanding protein translocation use biochemical systems in which spatial relationships are lost. 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 analyzed 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 have 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. COLLABORATORS Christopher Akey, PhD, Boston University School of Medicine, Boston MA a Left NICHD December 2002 For further information, contact hegder@mail.nih.gov |
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