Roles of SAM and DDHD domains in mammalian intracellular phospholipase A1 KIAA0725p

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Abstract

Members of the intracellular phospholipase A1 family of proteins have been implicated in organelle biogenesis and membrane trafficking. The mammalian family comprises three members: phosphatidic acid-preferring phospholipase A1 (PA-PLA1)/DDHD1, p125/Sec23ip and KIAA0725p/DDHD2, all of which have a DDHD domain. PA-PLA1 is mostly cytosolic, while KIAA0725p and p125 are more stably associated with the Golgi/endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) and ER exit sites, respectively. Here we show that KIAA0725p and p125 are novel phosphoinositide-binding proteins. Deletion and mutational analyses of KIAA0725p suggested that a sterile alpha-motif (SAM), which is also present in p125, but not in cytosolic PA-PLA1, and the following DDHD domain comprise a minimal region for phosphatidylinositol 4-phosphate (PI(4)P)-binding. A construct with mutations in the positively charged cluster of the SAM domain is defective in both phosphoinositide-binding and Golgi/ERGIC targeting. Consistent with the view that the PI(4)P-binding is important for the membrane association of KIAA0725p, expression of phosphoinositide phosphatase Sac1 reduces the association of expressed KIAA0725p with membranes. In addition, we show that deletion of the DDHD domain or introduction of point mutations at the conserved aspartate or histidine residues in the domain abolishes the phospholipase activity of KIAA0725p and PA-PLA1. Together, our results suggest that KIAA0725p is targeted to specific organelle membranes in a phosphoinositide-dependent manner, and that its SAM and DDHD domains are essential for its phosphoinositide-binding and phospholipase activity.

Highlights

► KIAA0725p and p125 are novel phosphoinositides-binding proteins. ► The SAM-DDHD domain in KIAA0725p is a minimal region for PI(4)P-binding. ► A positive charge cluster in the SAM domain is responsible for PI(4)P-binding. ► The lipid binding is necessary for targeting of KIAA0725p to Golgi/ERGIC membranes. ► The DDHD domain in KIAA0725p and PA-PLA1 is essential for their PLA1 activities.

Abbreviations

DOPA
1,2-dioleoyl-sn-phosphatidic acid
ER
endoplasmic reticulum
ERGIC
endoplasmic reticulum-Golgi intermediate compartment
GST
glutathione-S-transferase
iPLA1
intracellular phospholipase A1
LPA
1-oleoyl-lysophosphatidic acid
LUV
large unilamellar vesicles
PA
phosphatidic acid
PC
phosphatidylcholine
PE
phosphatidylethanolamine
PH
pleckstrin homology
PI(3)P
phosphatidylinositol 3-phosphate
PI(4)P
phosphatidylinositol 4-phosphate
PI(4,5)P2
phosphatidylinositol 4,5-bisphosphate
PI(5)P
phosphatidylinositol 5-phosphate
PI
phosphatidylinositol
PIP
phosphatidylinositol phosphate
PLA1
phospholipase A1
PS
phosphatidylserine
SAM
sterile alpha-motif
TLC
thin-layer chromatography
WT
wild-type

Keywords

KIAA0725p
Phospholipase A1
Phosphoinositide
Phosphatidylinositol 4-phosphate
SAM domain
DDHD domain

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This work was supported in part by Grants-in-Aid for Scientific Research 23570174 to H.I. and 20570190 to K.T. from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

1

These authors contributed equally to this work.