REGULATION OF SECRETORY AND MEMBRANE-PROTEIN BIOGENESIS
, Head, Unit on Protein Biogenesis
Ajay Sharma, PhD, Research Fellow
Martina Alken, PhD, Postdoctoral Fellow1
Aarthi Ashok, PhD, Postdoctoral Fellow
Oishee Chakrabarti, PhD, Postdoctoral Fellow
Sang-Wook Kang, PhD, Postdoctoral Fellow
Neena Rane, PhD, Postdoctoral Fellow2
Sandra Stefanovic, PhD, Postdoctoral Fellow3
Amy Emerman, BS, Postbaccalaureate Student
Heather Eshleman, BS, Postbaccalaureate Student4
Sanjay Bhat, Summer Student5
Amir Manoochehri, Summer Student4
Prion protein cell biology and neurodegeneration
The prion protein (PrP) is a widely expressed and highly conserved cell surface glycoprotein of uncertain function. Aberrant metabolism of PrP is responsible for a variety of neurodegenerative diseases in both humans and animals. These diseases include the transmissible “prion diseases” such as bovine spongiform encephalopathy as well as inheritable neurodegenerative diseases caused by mutations in the PrP gene. In neither case is the pathway(s) leading to cell death and neuronal damage understood. The overall goal of our project is to define the pathways of PrP-mediated neurodegeneration. To that end, we are studying the molecular pathways of PrP biosynthesis, intracellular trafficking, metabolism, and degradation. Quantitative analyses of these events are expected to shed light on how the various inherited mutations in PrP influence its biosynthesis or metabolism in a manner that leads to cellular dysfunction.
Our analysis suggests that at least two cytotoxic forms of PrP (termed CtmPrP and cyPrP) are made during the initial translocation of PrP into the ER. We have created transgenic mice to determine whether CtmPrP- and cyPrP-mediated neurodegeneration can be averted in vivo by modulating this newly discovered translocation during PrP biogenesis. To determine whether modulation of initial translocation 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 to discover 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 be able to slow or reverse the neurodegenerative process in these diseases.
In parallel, we are performing a systematic analysis of the biosynthesis, trafficking, and metabolism of disease-associated PrP mutants. The aim of our studies is to identify precisely the cellular locale and mechanism of PrP misfolding that initiate the disease process. Our current analyses have established that, for a large number of mutants, misfolded PrP is found in a post–ER location. This observation is notable because it suggests that the misfolded PrP species have somehow escaped the normal cellular quality control mechanisms in the ER. We are also studying the downstream consequences of PrP misfolding and aggregation to identify the mechanism by which these events lead to cellular dysfunction. We have now found that these aggregates recruit various cellular factors, thereby depleting their functional availability. One such factor is of particular importance because its disruption in mice leads directly to a neurodegenerative phenotype reminiscent of diseases caused by PrP.
Ashok A, Hegde RS. Prions and retroviruses: an endosomal rendezvous? EMBO Rep 2006;7:685-7.
Hegde RS, Chakrabarti O. Trafficking of the cellular prion protein and its role in neurodegeneration. In: Bean A, ed. Protein Trafficking in the Neuron. Elsevier, 2005;413-36.
Hegde RS, Rane NS. The molecular basis of prion protein-mediated neuronal damage. In: Brown DR, ed. Neurodegeneration and Prion Disease. Springer, 2005;407-50.
Kang SW, Rane NR, Kim SJ, Garrison JL, Taunton J, Hegde RS. Substrate-specific attenuation of protein translocation during acute ER stress defines a pathway of pre-emptive quality control. Cell 2006;127:999-1013.
Rutkowski DT, Kang SW, Goodman AG, Garrison JL, Taunton J, Katze MG, Kaufman RJ, Hegde RS. The role of p58IPK in protecting the stressed endoplasmic reticulum. Mol Biol Cell 2007;18:3681-91.
Regulation of protein biogenesis and function by signal sequences
Our discovery that the N-terminal signal sequence of PrP regulates PrP’s topology established a new function for PrP’s domain independent of its well-studied role in protein targeting. We subsequently developed and applied 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 the 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 revealed that many mutants are altered in their gating function rather than in their targeting function, as previously assumed. Thus, we have discovered that the long-observed sequence variations of signals do not simply represent functional degeneracy but also encode differences in translocon gating that are critical to the proper biogenesis of attached substrate. In a particularly dramatic example, we showed that conditions of ER stress attenuate the translocation of some but not other substrates in a signal sequence–dependent manner. This “pre-emptive” quality control (pQC) pathway is part of the adaptive cellular stress response and minimizes protein misfolding in the ER lumen. We are currently investigating whether the cell exploits substrate-specific properties of signal sequences in other ways as a means of regulating the subcellular localization of certain proteins, such as calreticulin, that have been shown to be present in several compartments. Our ongoing studies focus on developing a molecular understanding of the critical interaction between signal sequences and the translocon.
Alken M, Hegde RS. The translocation apparatus of the endoplasmic reticulum. In: Dalbey R, Koehler C, Tamanoi F, eds. Molecular Machines Involved in Protein Transport across Cellular Membranes. Academic Press/Elsevier, 2006;207-43.
Bernstein HD, Hegde RS. The surprising complexity of signal sequences. Trends Biochem Sci 2006;31:563-71.
Hegde RS. Protein translocation across the endoplasmic reticulum. In: Eichler J, ed. Protein Movement across Membranes. 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;16:279-91.
Shaffer KL, Sharma A, Snapp EL, Hegde RS. Regulation of protein compartmentalization expands the diversity of protein function. Dev Cell 2005;9:545-54.
Cryo–electron microscopy of the protein translocon
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 under different conditions such as that of a quiescent translocon, translocons engaged with a translocating substrate, and translocons lacking or containing individual components. To develop an understanding of the architecture of the protein translocation apparatus, we will fit the topography of these cryo–electron microscopy structures with atomic models of individual translocon structures obtained from existing and forthcoming crystal structures.
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;348:445-7.
The degradation machinery for mis-localized secretory and membrane proteins
What happens to copies of secretory and membrane proteins that fail to be properly segregated into the ER? While the simple answer is that they are degraded, a more precise answer delineating the pathway of their selective recognition, ubiquitination, and degradation is lacking. Given that these proteins are often markedly hydrophobic and/or contain unprocessed hydrophobic elements, they are at high risk for aggregation and inappropriate interactions. Thus, the pathway for the selective recognition and degradation of nontranslocated secretory and membrane proteins is likely to be of importance not only during pQC but also for normal cellular homeostasis. We are addressing this issue by using an in vitro system in which secretory and membrane proteins can be synthesized in their “nontranslocated” state by simply omitting ER-derived rough microsomes from the reaction (or, if the microsomes are included, by inhibiting translocation using Cotransin). With either of these manipulations, nontranslocated proteins are rapidly ubiquitinated in preparation for their degradation. We are employing both classical fractionation and affinity purification approaches to identify the machinery for the selective recognition and degradation of these nontranslocated proteins.
Small-molecule inhibitors of protein translocation
To develop new methods and probes for protein translocation, we have been developing pharmacologic methods of modulating translocation in vivo. We recently synthesized and characterized a novel small-molecule inhibitor of cotranslational protein translocation and demonstrated in both in vitro and cultured cells that this compound inihibits the translocation of some but not other proteins. Remarkably, we found substrate specificity to be encoded in the signal sequence. Thus, the sensitivity or resistance to the inhibitor can be conferred to any protein of interest simply by choice of signal. We have identified the target of this inhibitor as the Sec61 complex, the central component of the protein translocation channel. Our findings now open the way for selectively and potently modulating the translocation of individual substrates in live cells. We are applying our 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;436:285-9.
MacKinnon AL, Garrison JG, Hegde RS, Taunton J. Photo-leucine incorporation reveals the target of a cyclodepsipeptide inhibitor of cotranslational translocation. J Am Chem Soc 2007;129:14560-1.
Mechanism of tail-anchored protein insertion
Insertion of proteins into biological membranes is a fundamental process vital to all organisms. Most membrane proteins use the classical co-translational translocation pathway. The essential and universally conserved machinery for substrate recognition, targeting, and insertion by this pathway is well established and extensively characterized. By contrast, little is known about post-translational membrane protein insertion pathways. The main clients for this pathway are tail-anchored (TA) membrane proteins defined by a cytosolic-facing N-terminal domain followed by a single C-terminal transmembrane domain (TMD). Examples of TA proteins are found on essentially all cellular membranes in every organism; the proteins play diverse functional roles ranging from intracellular trafficking to regulation of cell death. Despite TA proteins’ widespread importance, the machinery and mechanisms underlying their recognition, targeting, and insertion into the correct organellar membrane remain largely unknown. Recently, we discovered a cytosolic TMD recognition complex (TRC) that selectively interacts with TA proteins destined for the ER membrane. We identified a central component of TRC as a highly conserved 40 kD ATPase that represents the first molecular factor in this widely used membrane protein insertion pathway. Other components that collaborate with TRC40 to mediate selective recognition, targeting, and insertion of TA proteins into the ER remain unknown. We are now taking various approaches to identifying these additional factor(s) and reconstituting the targeting reaction for TA proteins with defined components. Achieving these goals will define the core machinery and functions for a fundamental protein trafficking pathway, paving the way for future mechanistic and structural analyses.
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;24:2533-42.
Stefanovic S, Hegde RS. Identification of a targeting factor for post-translational membrane protein insertion into the ER. Cell 2007;128:1147-59.
1 Left NICHD, December 2006.2 Left NICHD, July 2007.
3 Left NICHD, October 2007.
4 Left NICHD, August 2007.
5 Left NICHD, August 2006.
COLLABORATORS
Christopher Akey, PhD, Boston University School of Medicine, Boston, MA
Lionel Feigenbaum, PhD, SAIC Frederick, NCI-Frederick Cancer Research and Development Center, Frederick, MD
Robert Keenan, PhD, University of Chicago, Chicago, IL
Jennifer Lippincott-Schwartz, PhD, Cell Biology and Metabolism Program, 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.
