MUTAGENESIS OF PHOSPHOLIPASE-D DEFINES A SUPERFAMILY INCLUDING A TRANS-GOLGI VIRAL PROTEIN REQUIRED FOR POXVIRUS PATHOGENICITY

Phospholipase D (PLD) genes are members of a superfamily that is defin ed by several highly conserved motifs, PLD in mammals has been propose d to play a role in membrane vesicular trafficking and signal transduc tion, Using site-directed mutagenesis, 25 point mutants have been made in human PLD1 (hPLD1) and characterized. We find that a motif (HxKxxx xD) and a serine/threonine conserved in all members of the PLD superfa mily are critical for PLD biochemical activity, suggesting a possible catalytic mechanism, Functional analysis of catalytically inactive poi nt mutants for yeast PLD demonstrates that the meiotic phenotype ensui ng from PLD deficiency in yeast derives from a loss of enzymatic activ ity. Finally, mutation of an HxKxxxxD motif found in a vaccinia viral protein expressed in the Golgi complex results in loss of efficient va ccinia virus cell-to-cell spreading, implicating the viral protein as a member of the superfamily and suggesting that it encodes a lipid mod ifying or binding activity. The results suggest that vaccinia virus an d hPLD1 may act through analogous mechanisms to effect viral cellular egress and vesicular trafficking, respectively.