The C2 domain of phosphatidylserine decarboxylase 2 is not required for catalysis but is essential for in vivo function.
Phosphatidylserine decarboxylase 2 (Psd2p) is currently being used to study lipid trafficking processes in intact and permeabilized yeast cells. The Psd2p contains a C2 homology domain and a putative Golgi retention/localization (GR) domain. C2 domains play important functions in membrane binding and docking reactions involving phospholipids and proteins. We constructed a C2 domain deletion variant (C2Delta) and a GR deletion variant (GRDelta) of Psd2p and examined their effects on in vivo function and catalysis. Immunoblotting confirmed that the predicted immature and mature forms of Psd2(C2Delta)p, Psd2(GRDelta)p, and wild type Psd2p were produced in vivo and that the proteins localized normally. Enzymology revealed that the Psd2(C2Delta)p and Psd2(GRDelta)p were catalytically active and could readily be expressed at levels 10-fold higher than endogenous Psd2p. Both Psd2p and Psd2(GRDelta)p expression complemented the growth defect of psd1Deltapsd2Delta strains and resulted in normal aminoglycerophospholipid metabolism. In contrast, the Psd2(C2Delta)p failed to complement psd1Deltapsd2Delta strains, and [(3)H]serine labeling revealed a severe defect in the formation of PtdEtn in both intact and permeabilized cells, indicative of disruption of lipid trafficking. These findings identify an essential, non-catalytic function of the C2 domain of Psd2p and raise the possibility that it plays a direct role in membrane docking and/or PtdSer transport to the enzyme.
Kitamura
Hidemitsu
H
Program in Cell Biology, Department of Medicine, National Jewish Medical and Research Center, Denver, CO 80206, USA.
Wu
Wen-I
WI
Voelker
Dennis R
DR
D000595:Amino Acid Sequence; D002262:Carboxy-Lyases / Q000737:chemistry* / Q000502:physiology; D020134:Catalytic Domain; D008969:Molecular Sequence Data; D010714:Phosphatidylethanolamines / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology
D010714:Phosphatidylethanolamines; D010718:Phosphatidylserines; D002262:Carboxy-Lyases; C022353:phosphatidylserine decarboxylase