Export of a misprocessed GPI-anchored protein from the endoplasmic reticulum in vitro in an ATP- and cytosol-dependent manner

Strict quality control mechanisms within the mammalian endoplasmic reticulu m act to prevent misfolded and unprocessed proteins from entering post-endo plasmic reticulum (ER) compartments. Following translocation into the ER lu men via the Sec61p translocon, nascent polypeptide chains fold and are modi fied in an environment that contains numerous chaperones and other folding mediators. Recently it has emerged that polypeptides failing to acquire the native state are re-exported from the ER to the cytosol for ultimate degra dation by the proteasome ubiquitin system, apparently mediated again via Se c61p. Substrates for this degradation pathway include proteins destined to become glycosyl phosphatidylinositol (GPI)-anchored, but which fail to be p rocessed and retain the C-terminal GPI signal peptide. In order to characte rise this process we have used a model GPI-anchored mutant protein, prepro mini human placental alkaline phosphatase (PLAP) W179, which cannot be proc essed efficiently on account of being a poor substrate for the transamidase which cleaves the GPI signal peptide and adds the GPI anchor in a coupled reaction. In vitro transcription, translation and translocation into canine pancreatic microsomes resulted in ER-targeting signal sequence cleavage an d formation of prominiPLAP in the ER lumen. We were able to show that promi niPLAPW179 could be exported from the microsomes in a time-dependent manner and that release requires both ATP and cytosol, Export was not supported b y GTP, indicating a biochemical distinction from glycopeptide export which we showed recently requires GTP hydrolysis. The process was not affected by redox, unlike several other GPI-anchored model proteins. These data demons trate that misprocessed proteins can be exported in vitro from mammalian mi crosomes, facilitating identification of factors involved in this process. (C) 2000 Federation of European Biochemical Societies. Published by Elsevie r Science B.V. All rights reserved.