We are interested in the molecular characterization of a quality control system that operates in the endoplasmic reticulum (ER) to ensure that only properly folded proteins will be released.  Misfolded polypeptides are retro-translocated from the ER to the cytosol, where they become poly-ubiquitinated and destructed by proteasomes.  ER-associated degradation (ERAD) is of relevance for a variety of genetically inherited, neurodegenerative, and virally transmitted diseases with protein folding defects.

We have previously shown that a truncated form of ribophorin I, a model glycoprotein for ERAD, is degraded by the ubiquitin/proteasome system. The role of N-linked glycans in ERAD was pinpointed as temporary retention devices in the ER. Thus, interaction of N-glycosylated substrates with the calnexin cycle appears to prolong their half lives. Furthermore, the requirement of N-linked glycan trimming for ERAD was shown, and from studies with mutant cell lines with defects in N-glycan assembly the activities of one or more ER α1,2-mannosidases could be implicated in ERAD.

Interaction partners of ERAD substrate proteins in these mutant cell lines will be determined in immunoprecipitation experiments with cell lysates from cells grown in the presence of proteasome and/or glycan processing inhibitors. Positive candidates will be identified using antibodies to known ER-associated proteins, and/or by mass spectroscopy and then further characterized. Another aspect of this project deals with the precise intracellular localization of the ERAD pathway of glycoproteins by indirect immunofluorescence and confocal laser scanning microscopy using appropriate marker proteins.

The role of MTP and PDI in the assembly and secretion of atherogenic lipoprotein particles

Microsomal triglyceride transfer protein (MTP) is a lipid transfer protein required for the assembly and secretion of very low density lipoproteins (VLDL). Active MTP is a heterodimer containing a 97 kDa catalytic subunit and a 58 kDa subunit identified as protein disulfide isomerase (PDI). The MTP complex catalyzes the loading of apolipoprotein B (apoB) with lipids and/or the translocation of apoB into the lumen of the endoplasmic reticulum (ER). In avians, the synthesis of VLDL is inducible by estrogen. We are studying the effect of estrogen treatment on MTP activity and on the regulation of VLDL secretion that is also determined by lipid availability and apoB degradation. In this context, the consequence of altered intracellular MTP activity on VLDL assembly and secretion is being analyzed. Another aspect of the project is concerned with the mechanism of retention of the MTP complex in the ER.