Biogenesis Of Secretory And Membrane Proteins

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The Unit on Protein Biogenesis studies 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 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, hypotheses regarding the molecular basis of particular diseases of the early secretory pathway are being formulated and tested in vivo. 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. Previous studies have established that 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 sec-PrP), and two transmembrane forms that span the membrane in opposite orientations (Ntm-PrP and Ctm-PrP). In vivo studies have revealed that even a slight overrepresentation of the Ctm-PrP topologic form results in the development of neurodegenerative disease in both mouse model systems and naturally occurring human disease. During the past year, our efforts have been focused on dissecting the mechanisms that direct an initially homogeneous cohort of nascent PrP polypeptides into multiple topologic forms to gain insight into not only the mechanisms underlying certain forms of prion disease, but also the basic mechanisms of secretory and membrane protein biogenesis. 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 step could be experimentally tuned to modify PrP topogenesis, including complete reversal of the elevated Ctm-PrP caused by disease-associated mutations in the transmembrane domain. Studies in transgenic mice are now being initiated to determine whether Ctm-PrP-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.