Ramanujan S. Hegde, MD, PhD, Head, Unit on Protein Biogenesis
Ajay Sharma, PhD, Research Fellow
Martina Alken, PhD, Postdoctoral Fellow
Aarthi Ashok, PhD, Postdoctoral Fellow
Oishee Chakrabarti, PhD, Postdoctoral Fellow
Sang-Wook Kang, PhD, Postdoctoral Fellow
Neena Rane, PhD, Postdoctoral Fellow
Sandra Stefanovic, PhD, Postdoctoral Fellow
Sanjay Bhat, Summer Student
Amir Manoochehri, Summer Student

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 include the import of secretory proteins, topogenesis and assembly of complex multispanning membrane proteins, and the export of misfolded substrates from the ER to the cytosol for degradation. Still unknown, however, are the mechanisms that allow the shared translocon to accommodate such an extensive range of substrates both efficiently and accurately. The principal goal of our studies is to define molecular mechanisms and components of the translocon that recognize the information in the primary sequence of the translocon's substrates and mediate 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 can formulate hypotheses regarding the molecular basis of particular diseases of the early secretory pathway and test them in vivo.

Prion protein cell biology and neurodegeneration

Rane, Chakrabarti, Ashok, Hegde N,¹ Manoochehri, Hegde R; in collaboration with Feigenbaum

The prion protein (PrP), a brain glycoprotein involved in various neurodegenerative diseases, has proven itself 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, the biogenesis of PrP 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 humans. 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 of the potentially harmful forms of PrP (CtmPrP and cyPrP) is the signal sequence-mediated translocation of the N-terminus of PrP into the ER lumen. We have demonstrated a constitutive, highly efficient signal sequence that can substantially reduce the generation of CtmPrP and cyPrP. The consequences of this manipulation in a cultured neuronal cell line are a marked reduction in 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 their initial translocation into the ER. We have created transgenic mice to determine whether CtmPrP-mediated neurodegeneration and cyPrP-mediated neurodegeneration can be averted in vivo by modulating this newly discovered step during PrP biogenesis. To determine whether such modulation is involved in the progression of neurodegeneration, we are also investigating the pathways by which the various forms of PrP are normally metabolized by the cell. We expect that a combination of defects in biosynthesis and/or clearance of certain forms of PrP collaborate to cause eventual neuronal dysfunction and death. Conversely, manipulation of these events may slow or reverse the neurodegenerative process in PrP-caused diseases.

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 have 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.

Regulation of protein biogenesis and function by signal sequences

Kang, Sharma, Levine,² Eagan ³; in collaboration with Sharan, Tessarollo

Our discovery that the N-terminal signal sequence of PrP regulates the protein'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 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 the mutants are altered in their gating function and, contrary to a previous assumption, do not target. Thus, we have discovered that the long-observed sequence variations in signals do not simply represent functional degeneracy but instead encode differences in translocon gating that are critical to the proper biogenesis of the translocon's attached substrate. We are currently investigating whether the cell exploits the substrate-specific properties of signal sequences in order to regulate the subcellular localization of certain proteins, such as calreticulin, that are present in several compartments. Furthermore, we are identifying conditions, such as ER stress, under which the cell adaptively modulates the translocation of some but not other substrates. By using RNAi-based screens in cell culture, we are investigating the mechanism of and factors involved in such regulation.

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 this 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 key component of the translocation machinery that aids in decoding the substrate-specific information in signal sequences. Our current studies of TRAP function use two approaches to help eludicate the molecular mechanism by which TRAP facilitates signal recognition, substrate translocation, and protein topogenesis. In collaboration with Christopher Akey's laboratory, 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 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 structural information combined with functional analyses will provide insight into the mechanisms by which TRAP can regulate protein translocation in a substrate-specific manner.

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 to modulate the translocation process in vivo. We recently synthesized and characterized a novel small-molecule inhibitor of co-translational protein translocation. Both in vitro and in cultured cells, the compound inhibits the translocation of some but not other proteins. Remarkably, we discovered that this substrate specificity was encoded in the signal sequence. Thus, the sensitivity or resistance to the inhibitor can be conferred to any protein of interest simply by the choice of signal. We identified the likely target of the inhibitor, which appears to be the Sec61 complex, the central component of the protein translocation channel. Our methods and findings now open the way to selectively and potently modulate the translocation of individual substrates in live cells. We are now applying this approach to study the role of protein translocation in various cellular events such as protein aggregation and toxicity, protein degradation, and the cellular response to ER stress.

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 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 not know whether all these translocon-associated activities are distributed homogeneously throughout the ER or organized and regulated in the spatial and temporal dimensions to meet the cell's changing needs. At present, there is little or no insight into this issue, 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 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 during cellular metabolism, development, or disease pathogenesis.

Mechanism of tail-anchored protein insertion

Stefanovic, Bhat; 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 inserting the lipid bilayer. Such tail-anchored (TA) proteins are found on all intracellular membranes exposed to the cytosol and are involved in a remarkably diverse range of physiologic processes, ranging from intracellular trafficking to protein degradation and 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. However, 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. We found that for one model protein, cytochrome b5, protein factors are not obligatorily involved. Instead, insertion appears to be influenced by the lipid composition of the target membrane, with at least one factor being cholesterol. By contrast, other model TA proteins appear to require protein factors in both the cytosol and target membrane. We are now identifying these factors by using fractionation and functional reconstitution approaches.

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 constitute themselves properly with high fidelity. This is a particularly important issue because multispanning proteins include some of the most important cell surface molecules, such as ion channels, transporters, and receptors, that are defective in many diseases and therefore are targets for many therapeutic drugs. Issues to be resolved include the feasibility of functionally reconstituting the process of multispanning protein formation in a biochemical system amenable to fractionation; the functional requirements for the formation of such proteins; and the steps at which the proteins' biosynthesis is defective in disease-associated mutants of such membrane proteins. We are using several G protein-coupled receptors as model membrane proteins to begin addressing these issues. We have thus far reconstituted the biosynthesis and insertion of at least some receptors in an in vitro system and are using the manipulability of the system to identify factors involved in key biosynthetic steps. We expect that these efforts will provide critical insight into the mechanisms of membrane protein assembly and the consequences of dysregulation of these events for disease pathogenesis.

¹ Niyathi Hegde, former Summer Student, left NICHD in August 2005.
² Corinna Levine, BS, former Postbaccalaureate Fellow, left NICHD in August, 2004.
³ Carolyn Eagan, former Summer Student, left NICHD in August 2005.

Collaborators

Christopher Akey, PhD, Boston University School of Medicine, Boston, MA
Nica Borgese, PhD, Consiglio Nazionale delle Recerche, Milan, Italy
Lionel Feigenbaum, PhD, SAIC, 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.