Cell Lines from Kidney Proximal Tubules of a Patient with Lowe Syndrome Lack OCRL Inositol Polyphosphate 5-Phosphatase and Accumulate Phosphatidylinositol 4,5-Bisphosphate

Xiaoling Zhang,Patricia A Hartz,Elizabeth Philip,Lorraine C Racusen,Philip W Majerus

doi:10.1074/jbc.273.3.1574

Abstract

The protein product of the gene that when mutated is responsible for Lowe syndrome, or oculocerebrorenal syndrome (OCRL), is an inositol polyphosphate 5-phosphatase. It has a marked preference for phosphatidylinositol 4,5-bisphosphate although it hydrolyzes all four of the known inositol polyphosphate 5-phosphatase substrates: inositol 1,4,5-trisphosphate, inositol 1,3,4,5-tetrakisphosphate, phosphatidylinositol 4,5-bisphosphate, and phosphatidylinositol 3,4,5-trisphosphate. The enzyme activity of this protein is determined by a region of 672 out of a total of 970 amino acids that is homologous to inositol polyphosphate 5-phosphatase II. Cell lines from kidney proximal tubules of a patient with Lowe syndrome and a normal individual were used to study the function of OCRL. The cells from the Lowe syndrome patient lack OCRL protein. OCRL is the major phosphatidylinositol 4,5-bisphosphate 5-phosphatase in these cells. As a result, these cells accumulate phosphatidylinositol 4,5-bisphosphate even though at least four other inositol polyphosphate 5-phosphatase isozymes are present in these cells. OCRL is associated with lysosomal membranes in control proximal tubule cell lines suggesting that OCRL may function in lysosomal membrane trafficking by regulating the specific pool of phosphatidylinositol 4,5-bisphosphate that is associated with lysosomes.

Highlights

The protein product of the gene that when mutated is responsible for Lowe syndrome, or oculocerebrorenal syndrome (OCRL), is an inositol polyphosphate 5-phosphatase
OCRL is associated with lysosomal membranes in control proximal tubule cell lines suggesting that OCRL may function in lysosomal membrane trafficking by regulating the specific pool of phosphatidylinositol 4,5-bisphosphate that is associated with lysosomes
It appears that the 5-phosphatase II homology region of OCRL determines its enzyme activity, whereas the amino-terminal region of OCRL is not required for its enzyme activity

Summary

EXPERIMENTAL PROCEDURES

Materials—[3H]Inositol, [3H]Ins1,3,4,5-P4, [3H]PtdIns 4,5-P2, and [␥-32P]ATP were purchased from NEN Life Science Products. The polymerase chain reaction product obtained contains amino acids Met to Met506 of OCRL1 It was digested with BamHI/SphI and subcloned into the same region of ps.OCRLB-1 to replace amino acids 1–506 of the ps.OCRLB-1 cDNA. The cell extracts were fractionated by eluting the column at 1 ml/min with buffer B (buffer A containing 1 M NaCl) programmed as follows: 0 –10 min, buffer A (flow-through); 10 –30 min, 0 –50% B; 30 –35 min, 50 –100% B. The cells were incubated with a mixture of primary antibodies including anti-OCRL polyclonal antibody and anti␥-adaptin monoclonal antibody, or anti-OCRL polyclonal antibody and anti-protein disulfate isomerase monoclonal antibody, or anti-OCRL polyclonal antibody and anti-lamp monoclonal antibody, respectively This first incubation was followed by a second incubation with a mixture of secondary antibodies including fluorescein isothiocyanate-conjugated donkey anti-rabbit IgG and indocarbocyanine (Cy3)-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch). Slides were observed using a Nikon microscope system (ECLIPSE E800)

RESULTS

TABLE I

LS cells cpm PtdIns

DISCUSSION