V-ATPase is a major component of the Golgi complex in the scaly green flagellate Scherffelia dubia

Highly purified membranes isolated from the Golgi complex of the scaly gree n flagellate Scherffelia dubia (Chlorophyta) were subjected to Triton X-114 two-phase partitioning. Proteins in the detergent phase were analyzed by 2 D gel electrophoresis and a major protein of 66 kD (p66) was N-terminally s equenced. The complete cDNA sequence of p66 was obtained by 3' RACE-PCR and screening of a cDNA library of S. dubia with a PCR probe derived from the 3' RACE. Sequence analysis of the cDNA clone identified p66 as subunit A of V-ATPase. Other major proteins in the isolated Golgi complex were immunore active to heterologous antibodies raised against subunit B or the holoenzym e of V-ATPase. A polyclonal (anti-p66) antibody raised against a recombinan t, bacterially expressed p66 fusion protein recognized p66 in the isolated Golgi complex in western blots and localized the antigen by immunogold elec tron microscopy mostly to the scale reticulum but also to the Golgi stack w ithin the Golgi complex. Concanamycin A-sensitive (but bafilomycin A(1)-ins ensitive) ATPase activity was present in the isolated Golgi complex, and mo nensin at 0.5-1 mu M reversibly inhibited flagellar regeneration and result ed in swelling of Golgi cisternae. It is concluded that a functional V-ATPa se is a major protein of the Golgi complex in S. dubia and is presumably as sociated with sorting processes at the endocytotic/exocytotic boundary of t he Golgi complex.