Our previous studies have demonstrated that alterations in the initial translocation of the Prion protein (PrP) into the endoplasmic reticulum (ER) can lead to the development of neurodegenerative disease. During the past year, my group has made significant progress towards not only providing a molecular description of PrP translocation, but demonstrating how key steps during this process can be modulated to influence the generation of potentially neurotoxic forms of PrP. In particular, we have discovered that the most important and tightly regulated step in PrP biogenesis is the interaction between its signal sequence and the protein translocon. This step was found to be critically dependent on a four protein complex of previously unknown function (termed the TRAP complex), in the absence of which PrP does not enter the ER. Our finding that not all signal sequences require TRAP suggests that different substrates are recognized differently by the translocon, an idea further supported by recent studies analyzing crosslinking between signal sequences and translocon components. More significantly, we have now shown that alterations in the nature of this signal-translocon interaction have substantial consequences for protein localization and function. In the case of PrP, the cellular burden of potentially cytotoxic forms can be reduced (or enhanced) to change the susceptibility of cells to otherwise harmful insults. In the case of another protein, Calreticulin, we find that signal-translocon interactions are critical in allowing this protein to exist in two compartments (the ER lumen and the cytosol), where it serves independent functions. Thus, advances during the past year are beginning to illuminate a novel site of potential cellular regulation, the entry of secretory and membrane protein substrates into the mammalian secretory pathway, that impacts both normal physiology and disease progression. In parallel collaborative studies, we are using both structural and pharmacological approaches to understand components of the protein translocation machinery at the mammalian ER. In the structural approach, we are applying cryo-electron microscopy to visualize intact ribosome-translocon complexes. By preparing and analyzing translocon complexes lacking or containing specific components such as the TRAP complex, we are able to determine the relative positions of the various proteins comprising the translocon. In the pharmacologic approach, we are utilizing novel assays for translocation to identify, characterize, and study small molecule inhibitors of protein translocation. The goal of these studies is to develop probes that facilitate the modulation of protein translocation in vivo to understand the role of this process in normal and pathological cellular physiology.

Project Start
Project End
Budget Start
Budget End
Support Year
3
Fiscal Year
2004
Total Cost
Indirect Cost
Name
U.S. National Inst/Child Hlth/Human Dev
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Shao, Sichen; Hegde, Ramanujan S (2011) A calmodulin-dependent translocation pathway for small secretory proteins. Cell 147:1576-88
Stefanovic, Sandra; Hegde, Ramanujan S (2007) Identification of a targeting factor for posttranslational membrane protein insertion into the ER. Cell 128:1147-59
Ong, Hwei L; Liu, Xibao; Sharma, Ajay et al. (2007) Intracellular Ca(2+) release via the ER translocon activates store-operated calcium entry. Pflugers Arch 453:797-808
Ong, Hwei Ling; Liu, Xibao; Tsaneva-Atanasova, Krasimira et al. (2007) Relocalization of STIM1 for activation of store-operated Ca(2+) entry is determined by the depletion of subplasma membrane endoplasmic reticulum Ca(2+) store. J Biol Chem 282:12176-85
Rutkowski, D Thomas; Kang, Sang-Wook; Goodman, Alan G et al. (2007) The role of p58IPK in protecting the stressed endoplasmic reticulum. Mol Biol Cell 18:3681-91
Ashok, Aarthi; Hegde, Ramanujan S (2006) Prions and retroviruses: an endosomal rendezvous? EMBO Rep 7:685-7
Hegde, Ramanujan S; Bernstein, Harris D (2006) The surprising complexity of signal sequences. Trends Biochem Sci 31:563-71
Kang, Sang-Wook; Rane, Neena S; Kim, Soo Jung et al. (2006) Substrate-specific translocational attenuation during ER stress defines a pre-emptive quality control pathway. Cell 127:999-1013
Snapp, Erik L; Sharma, Ajay; Lippincott-Schwartz, Jennifer et al. (2006) Monitoring chaperone engagement of substrates in the endoplasmic reticulum of live cells. Proc Natl Acad Sci U S A 103:6536-41
Menetret, Jean-Francois; Hegde, Ramanujan S; Heinrich, Sven U et al. (2005) Architecture of the ribosome-channel complex derived from native membranes. J Mol Biol 348:445-57

Showing the most recent 10 out of 26 publications