Ocrl1, a PtdIns(4,5)P₂5-Phosphatase, Is Localized to theTrans-Golgi Network of Fibroblasts and Epithelial Cells

Abstract

SUMMARY PtdIns(4,5)P 2 and PtdIns(4,5)P 2 5-phosphatases play important roles in diverse aspects of cell metabolism, including protein trafficking. However, the relative importance of the PtdIns(4,5)P 2 5-phosphatases in regulating PtdIns(4,5)P 2 levels for specific cell processes is not well understood. Ocrl1 is a PtdIns(4,5)P 2 5-phosphatase that is deficient in the oculocerebrorenal syndrome of Lowe, a disorder characterized by defects in kidney and lens epithelial cells and mental retardation. Ocrl1 was originally localized to the Golgi in fibroblasts, but a subsequent report suggested a lysosomal localization in a kidney epithelial cell line. In this study we defined the localization of ocrl1 in fibroblasts and in two kidney epithelial cell lines by three methods: immunofluorescence, subcellular fractionation, and a dynamic perturbation assay with brefeldin A. We found that ocrl1 was a Golgi-localized protein in all three cell types and further identified it as a protein of the trans -Golgi network (TGN). The TGN is a major sorting site and has the specialized function in epithelial cells of directing proteins to the apical or basolateral domains. The epithelial cell phenotype in Lowe syndrome and the localization of ocrl1 to the TGN imply that this PtdIns(4,5)P 2 5-phosphatase plays a role in trafficking.

Targeting of the type II inositol polyphosphate 5-phosphatase INPP5B to the early secretory pathway

“…By contrast, under similar conditions, GFP-OCRL1 overlapped best with TGN46 (see supplementary material Fig. S1), consistent with an enrichment on the TGN, as suggested by previous work (Dressman et al, 2000). Localisation of INPP5B to the Golgi was not a fixation artefact and was observed using both methanol-and paraformaldehyde-fixed cells (supplementary material Fig.…”

“…However, again, there are notable differences. INPP5B binds to RAB2, whereas OCRL1 does not, and our immunofluorescence data suggest that INPP5B is enriched towards the cis-side of the Golgi apparatus, with OCRL1 more abundant at the trans side (see also Dressman et al, 2000). INPP5B is more abundant on the ERGIC than OCRL1, and its expression inhibits retrograde ERGIC-to-ER trafficking of ERGIC53, whereas expression of OCRL1 does not.…”

Phosphoinositides in Constitutive Membrane Traffic

“…In mammals there are a number of other 5-phosphatases, most of which have been investigated for roles in signal transduction or apoptosis, but not membrane traffic. However, the OCRL gene product that is defective in the human disease Lowe syndrome is a 5-phosphatase that localizes to the TGN (87,344,408). Kidney cells from patients with Lowe syndrome have elevated PtdIns(4,5)P 2 levels (407) as well as elevated serum levels of lysosomal enzymes (360), raising the possibility that there is a defect in sorting proteins at the TGN.…”

All known patient mutations in the ASH-RhoGAP domains of OCRL affect targeting and APPL1 binding

“…OCRL was initially localized to the Golgi complex [9,10]. Recently, OCRL was also found to have a prominent localization on endosomes and to be important at early stations of the endocytic pathway, including clathrincoated pits [11], consistent with its ability to bind clathrin, the endocytic clathrin adaptor AP-2, the endosomal protein Rab5, and the plasma membrane associated Rho family GTPases Rac and Cdc42 [12][13][14][15].…”