Soluble constituents of the ER lumen are required for GPI anchoring of a model protein.
Transfer of a glycosylphosphatidylinositol (GPI) anchor to proteins carrying a C-terminal GPI-directing signal sequence occurs after protein translocation across the endoplasmic reticulum (ER). We describe the translocation and GPI modification of a model protein, preprominiPLAP, in ER microsomes depleted of lumenal content by high pH washing. In untreated microsomes preprominiPLAP was processed to prominiPLAP and GPI-anchored miniPLAP. Both products were fully translocated, since they resisted proteinase K treatment of the microsomes, and both behaved as membrane proteins by the carbonate extraction criterion. Microsomes depleted of lumenal content were able to translocate and process preprominiPLAP to give protease-protected prominiPLAP, but were unable to convert prominiPLAP to miniPLAP. Loss of GPI anchoring capacity occurred with a wash of pH > 9.5. If the alkaline wash was performed after formation of prominiPLAP conversion to miniPLAP was relatively unimpaired. The results indicate that constituents of the ER lumen, possibly chaperones interacting with the proprotein and/or the GPI anchor precursor, are required in the initial steps of GPI anchoring.
Vidugiriene
J
J
Department of Biochemistry, University of Wisconsin-Madison 53706-1569, USA.
Menon
A K
AK
D000255:Adenosine Triphosphate / Q000502:physiology; D000469:Alkaline Phosphatase / Q000378:metabolism*; D000818:Animals; D019286:Endopeptidase K; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000502:physiology; D006863:Hydrogen-Ion Concentration; D051379:Mice; D008861:Microsomes / Q000378:metabolism; D010920:Placenta / Q000201:enzymology; D011388:Prolactin / Q000378:metabolism; D011498:Protein Precursors / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D012697:Serine Endopeptidases / Q000378:metabolism; D013945:Thymoma; D014407:Tumor Cells, Cultured
D017261:Glycosylphosphatidylinositols; D011498:Protein Precursors; C017671:preprolactin; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D011388:Prolactin; D000469:Alkaline Phosphatase; D012697:Serine Endopeptidases; D019286:Endopeptidase K