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 defined by several highly conserved motifs. PLD in mammals has been proposed to play a role in membrane vesicular trafficking and signal transduction. Using site-directed mutagenesis, 25 point mutants have been made in human PLD1 (hPLD1) and characterized. We find that a motif (HxKxxxxD) and a serine/threonine conserved in all members of the PLD superfamily are critical for PLD biochemical activity, suggesting a possible catalytic mechanism. Functional analysis of catalytically inactive point mutants for yeast PLD demonstrates that the meiotic phenotype ensuing from PLD deficiency in yeast derives from a loss of enzymatic activity. Finally, mutation of an HxKxxxxD motif found in a vaccinia viral protein expressed in the Golgi complex results in loss of efficient vaccinia virus cell-to-cell spreading, implicating the viral protein as a member of the superfamily and suggesting that it encodes a lipid modifying or binding activity. The results suggest that vaccinia virus and hPLD1 may act through analogous mechanisms to effect viral cellular egress and vesicular trafficking, respectively.
Sung
T C
TC
Department of Pharmacological Sciences, SUNY at Stony Brook, NY 11794-8651, USA.
Roper
R L
RL
Zhang
Y
Y
Rudge
S A
SA
Temel
R
R
Hammond
S M
SM
Morris
A J
AJ
Moss
B
B
Engebrecht
J
J
Frohman
M A
MA
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D019556:COS Cells; D002384:Catalysis; D017124:Conserved Sequence; D019143:Evolution, Molecular; D006801:Humans; D008239:Lysine / Q000235:genetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010739:Phospholipase D / Q000235:genetics*; D017354:Point Mutation; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014616:Vaccinia virus / Q000201:enzymology / Q000235:genetics* / Q000472:pathogenicity*; D014764:Viral Proteins / Q000235:genetics*
D014764:Viral Proteins; D010739:Phospholipase D; D008239:Lysine