PROTEIN FOLDING IN THE ENDOPLASMIC RETICULUM

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While protein folding has been extensively studied in vitro, little is known about the process in living cells. Our goal is to elucidate how glycoproteins fold within the lumen of the endoplasmic reticulum(ER), and how the outcome of the folding process determines their fate within the cell. Secretory proteins, membrane glycoproteins and vacuolar proteins, which fold in this compartment, encounter in the ER an environment which is different in many ways from any folding compartment in the cell where protein folding takes place. As model proteins, we will rely mainly on two well-characterized viral membrane glycoproteins; influenza hemagglutinin (HA) and vesicular stomatitis virus (VSV) G protein. Our main objectives are to determine to which extent the kinetics and efficiency of co- and post-translational folding depend on stress proteins, folding factors, chaperonins, signal sequence cleavage, N-linked glycosylation, redox potential, translation rate, calcium concentration and temperature. Since a large part of the folding is co-translational, we will determine whether the translation rate and ribosome pausing regulate the process. We will isolate and characterize new ER factors responsible for folding, misfolding and retention of proteins in the ER and in associated organelles. We will investigate the molecular signals that prevent misfolded proteins from exiting to the Golgi complex, and induce their degradation. The organelle where degradation occurs will be analyzed and isolated. To better understand the structure of the ER, we will finally study the composition and function of the matrix of insoluble proteins in the ER membrane and lumen. This matrix is likely to be involved in the quality control processes of the ER. By focussing on the cell biological aspects of the co- and post translational folding i living cells, we hope to learn more about the fundamental aspect of secretion, organelle biogenesis and post- translational regulation. Hopefully, the results will also throw some light on a number of pathological states which can be categorized as "ER storage diseases".