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

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-PIA1)/DDHD1, p125/Sec23ip and KIAA0725p/DDHD2, all of which have a DDHD domain. PA-PLAI 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 KIAAO725p and p125 are novel phosphoinositide-binding proteins. Deletion and mutational analyses of KIAAO725p suggested that a sterile alpha-motif (SAM), which is also present inp125, but not in cytosolic PA-PLAI, and the following DDHD domain comprise a minimal region for phosphatidylinositol 4-phosphate (Pl(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 Pl(4)P-binding is important for the membrane association of KIAA0725p, expression of phosphoinositide phosphatase Sacd reduces the association of expressed KIAAO725p 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 KIAAO725p and PA-PLA1. Together, our results suggest that KIAAO725p 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.

Mutations in phospholipase DDHD2 cause autosomal recessive hereditary spastic paraplegia (SPG54)

“…[11][12][13] Overexpression of DDHD2 leads to dispersion of the Golgi and enlargement of the perinuclear ERGIC. 12,14 Conflicting data exists about the effect of DDHD2 depletion. Morikawa et al 13 report a specific defect of retrograde transport from the Golgi to the ER; this defect is not confirmed by Sato et al 11 who instead postulate a anterograde transport defect from the Golgi to the plasma membrane.…”

“…Catalytic function as well as a positively charged cluster in the SAM domain (Arg434-Lys435-Lys436) also required for phosphoinositide binding are necessary to promote membrane localization. 11,14 Mutations previously described 28 are indicated in black, novel mutations in red.…”

Mutations in CYP2U1, DDHD2 and GBA2 genes are rare causes of complicated forms of hereditary spastic paraparesis

“…Members of this family exhibit enzymatic activity and hydrolyse the acyl-group of phospholipids in the sn-1 position and share a conserved lipase motif (GxSxG) and a DDHD domain. This multifunctional domain along with the sterilealpha-motif (SAM) is essential for the phospholipase activity of the DDHD2 protein [18]. It is possible that DDHD2 plays a role by facilitating membrane and vesicle fusion through phospholipid hydrolysis.…”

Protein purification and cloning of diacylglycerol lipase from rat brain

“…It contains four conserved residues, Asp530, Asp648, His699 and Asp677. The domain is conserved in several phosphoesterases (22).…”

“…Because DG can potentially be produced in the membrane by PLC , membrane-bound DDHD2 might be a functional form in vivo. The binding of DDHD2 to membrane can be explained by its affinity to phosphoinositides (22). Because HA-rDDHD2 expressed in CHO cells exhibited unusual localization mainly in the cytosol (Fig.…”