Cloning and characterization of a 72-kDa inositol-polyphosphate 5-phosphatase localized to the Golgi network.
The inositol-polyphosphate 5-phosphatase enzyme family removes the 5-position phosphate from both inositol phosphate and phosphoinositide signaling molecules. We have cloned and characterized a novel 5-phosphatase, which demonstrates a restricted substrate specificity and tissue expression. The 3.9-kb cDNA predicts for a 72-kDa protein with an N-terminal proline rich domain, a central 5-phosphatase domain, and a C-terminal CAAX motif. The 3. 9-kilobase mRNA showed a restricted expression but was abundant in testis and brain. Antibodies against the sequence detected a 72-kDa protein in the testis in the detergent-insoluble fraction. Indirect immunofluorescence of the Tera-1 cell line using anti-peptide antibodies to the 72-kDa 5-phosphatase demonstrated that the enzyme is predominantly located to the Golgi. Expression of green fluorescent protein-tagged 72-kDa 5-phosphatase in COS-7 cells revealed that the enzyme localized predominantly to the Golgi, mediated by the N-terminal proline-rich domain, but not the C-terminal CAAX motif. In vitro, the protein inserted into microsomal membranes on the cytoplasmic face of the membrane. Immunoprecipitated recombinant 72-kDa 5-phosphatase hydrolyzed phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3, 5-bisphosphate, forming phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3-phosphate, respectively. We propose that the novel 5-phosphatase hydrolyzes phosphatidylinositol 3,4, 5-trisphosphate and phosphatidylinositol 3,5-bisphosphate on the cytoplasmic Golgi membrane and thereby may regulate Golgi-vesicular trafficking.
Kong
A M
AM
Department of Biochemistry and Molecular Biology and Department of Anatomy and Cell Biology, Monash University, Clayton, Victoria 3168, Australia.
Speed
C J
CJ
O'Malley
C J
CJ
Layton
M J
MJ
Meehan
T
T
Loveland
K L
KL
Cheema
S
S
Ooms
L M
LM
Mitchell
C A
CA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D001921:Brain / Q000201:enzymology; D002451:Cell Compartmentation; D016764:Cell Polarity; D003001:Cloning, Molecular; D005260:Female; D006056:Golgi Apparatus / Q000201:enzymology*; D017403:In Situ Hybridization; D000072181:Inositol Polyphosphate 5-Phosphatases; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D021382:Protein Sorting Signals; D012333:RNA, Messenger / Q000302:isolation & purification; D013379:Substrate Specificity; D013737:Testis / Q000201:enzymology; D014018:Tissue Distribution
D018129:Phosphatidylinositol Phosphates; D021382:Protein Sorting Signals; D012333:RNA, Messenger; C060974:phosphatidylinositol 3,4,5-triphosphate; C106336:phosphatidylinositol 3,5-diphosphate; D010744:Phosphoric Monoester Hydrolases; D000072181:Inositol Polyphosphate 5-Phosphatases