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 multi-spanning membrane proteins, to the export of misfolded substrates from the ER to the cytosol for degradation. The mechanisms that allow the shared translocon to accommodate such an extensive range of substrates efficiently yet accurately remain largely unknown. The principal goal of ongoing studies is to define the molecular mechanisms and components of the translocon that recognize the information in the primary sequence of its substrates to 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 are formulating and testing in vivo hypotheses regarding the molecular basis of particular diseases of the early secretory pathway.

Molecular Mechanism of Prion Protein Topogenesis
Kim
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 both in mouse-model systems and in the spontaneous forms of 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 depends critically on the precise timing of signal sequence–mediated initiation of N-terminus translocation. Consequently, the initiation 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 now 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 such 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 topology. Ongoing studies are investigating the role of these newly discovered factors in the biogenesis of other substrates (see below) and their potential role in the pathogenesis of PrP-associated neurodegeneration.

Regulation of Protein Biogenesis by Signal Sequences
Kim, Shaffer, Mitra
Our discovery that PrP topology is regulated by the N-terminal signal sequence has established a new function for this domain that is independent of the domain’s well-studied role in protein targeting. Using signal sequence mutants that uncouple targeting and post-targeting functions, we demonstrated that the post-targeting function lies in gating the translocation channel to provide the nascent polypeptide with access to the ER lumen. 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. Such substrate-specific properties of signals were found to be 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 have revealed that many mutants are altered in their gating function, and not in the targeting function, 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 proper biogenesis of its 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.

A Function for TRAP in Substrate-Specific Protein Translocation
Rane, Yonkovich, Hegde, Fons
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. The substrate-specificity proved to be 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 focus on under-standing the molecular mechanism by which it facilitates signal recognition, substrate trans-location, and protein topogenesis. In addition, we are investigating the consequence in vivo of RNAi-mediated suppression of TRAP expres-sion in cultured cells.

The Role of ER Chaperones in PrP Topogenesis
Kim, Hegde, Fons
Cross-linking analysis of PrP translocation intermediates has identified several proteins that are presumably involved in some aspect of its biogenesis. By comparing cross-linking patterns for different topology-altering mutants of PrP, we were able to identify several lumenal chaperones, including PDI, as proteins that selectively cross-link to translocation intermediates of secPrP. Biochemical extraction of the lumenal contents from ER-derived microsomes resulted in membranes that preferentially generate the transmembrane forms of PrP, suggesting that lumenal chaperones are required for proper PrP translocation. We have recently discovered that, under conditions of ER stress in cultured cells, when lumenal chaperones are functionally depleted by unfolded substrates, newly synthesized PrP appears to be improperly translocated. Importantly, the transport of certain other substrates is not impaired, arguing against a global defect in translocation. Given that dysregulation of PrP topogenesis can lead to neurodegeneration, our findings suggest a link between cellular stress, PrP topology, and disease pathogenesis. We are currently investigating whether titration of ER chaperones during prion disease may play a role in neurodegeneration through the inappropriate topogenesis of PrP.

Visualization of Translocon Organization in Cells
Hegde, Reinhart, Schwartz; in collaboration with Snapp, Lippincott-Schwartz
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 multi-spanning 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 the spatial and temporal dimensions to meet the changing needs of the cell. There is presently 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 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 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, which 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 patho-genesis.