Biochemical and Biophysical Research Communications, Vol.512, No.3, 584-590, 2019
Stability of the transamidase complex catalyzing GPI anchoring of proteins
Glycosylphosphatidylinositol (GPI) is a glycolipid that anchors some proteins to the plasma membrane. This anchoring is catalyzed by a transamidase complex (TAC) composed of five subunits: PIG-K, GAA1, PIG-U, PIG-T, and PIG-S (Fig. 1A). P1G-K and GAA1 are predicted to catalyze the first and second steps during attachment of proproteins of GPI-anchored proteins (GPI-APs) to GPI. GPI may be delivered by PIG-U, and PIG-T is required for stability of all TAC subunits when overexpressed in cultured cells. However, protein stability of TAC has not been analyzed using loss-of-function mutants for each subunit. Herein, we analyzed the stability of TAC in knockout and/or knockdown mutants for each subunit. PIG-T and PIG-U, or PIG-T and GAA1, were mutually required for stability, and all three subunits were stable without PIG-S or PIG-K. However, these three subunits were essential for the stability of both PIG-S and PIG-K. By contrast, loss of PIG-S reduced the stability of PIG-K and left the other subunits unaffected. Reduction of PIG-K did not impact any of the other subunits. Thus, PIG-T, PIG-U, and GAA1 may form a core complex associated by PIG-S, and these four subunits may stabilize PIG-K, triggering GPI anchoring reactions. Instability of PIG-K in the absence of the other four subunits may ensure that GPI anchoring is catalyzed only by the completely assembled complex. (C) 2019 Elsevier Inc. All rights reserved.
Keywords:Glycosylphosphatidylinositol;Transamidation;Enzyme complex;Protein stability;Organelle zone;Drosophila