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

FEBS Letters

Field, Mark C¹  Claxton, Sheyi¹  Ali, Bassam R.S¹ 

[1] Centre for Molecular Microbiology and Infection, Wellcome Trust Laboratories for Molecular Parasitology, Department of Biochemistry, Imperial College of Science, Technology and Medicine, Exhibition Road, London SW7 2AY, UK

关键词: Misfolded protein;    Glycopeptide;    Glycosyl phosphatidylinositol;    Endoplasmic reticulum;    Endoplasmic reticulum-associated degradation;    Transport;    ERAD;    endoplasmic reticulum-associated degradation;    ER;    endoplasmic reticulum;    GPI;    glycosyl phosphatidylinositol;    mPLAP;    mini human placental alkaline phosphatase;   

DOI  :  10.1016/S0014-5793(00)02073-1

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Strict quality control mechanisms within the mammalian endoplasmic reticulum act to prevent misfolded and unprocessed proteins from entering post-endoplasmic reticulum (ER) compartments. Following translocation into the ER lumen via the Sec61p translocon, nascent polypeptide chains fold and are modified 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 degradation by the proteasome ubiquitin system, apparently mediated again via Sec61p. Substrates for this degradation pathway include proteins destined to become glycosyl phosphatidylinositol (GPI)-anchored, but which fail to be processed and retain the C-terminal GPI signal peptide. In order to characterise this process we have used a model GPI-anchored mutant protein, prepro mini human placental alkaline phosphatase (PLAP) W179, which cannot be processed 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 and formation of prominiPLAP in the ER lumen. We were able to show that prominiPLAPW179 could be exported from the microsomes in a time-dependent manner and that release requires both ATP and cytosol. Export was not supported by 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 demonstrate that misprocessed proteins can be exported in vitro from mammalian microsomes, facilitating identification of factors involved in this process.

【 授权许可】

Unknown