OCRL1 engages with the F-BAR protein pacsin 2 to promote biogenesis of membrane-trafficking intermediates.

Mutation of the inositol 5-phosphatase OCRL1 causes Lowe syndrome and Dent-2 disease. Loss of OCRL1 function perturbs several cellular processes, including membrane traffic, but the underlying mechanisms remain poorly defined. Here we show that OCRL1 is part of the membrane-trafficking machinery operating at the trans-Golgi network (TGN)/endosome interface. OCRL1 interacts via IPIP27A with the F-BAR protein pacsin 2. OCRL1 and IPIP27A localize to mannose 6-phosphate receptor (MPR)-containing trafficking intermediates, and loss of either protein leads to defective MPR carrier biogenesis at the TGN and endosomes. OCRL1 5-phosphatase activity, which is membrane curvature sensitive, is stimulated by IPIP27A-mediated engagement of OCRL1 with pacsin 2 and promotes scission of MPR-containing carriers. Our data indicate a role for OCRL1, via IPIP27A, in regulating the formation of pacsin 2-dependent trafficking intermediates and reveal a mechanism for coupling PtdIns(4,5)P2 hydrolysis with carrier biogenesis on endomembranes.

DNA damage triggers Golgi dispersal via DNA-PK and GOLPH3.

The response to DNA damage, which regulates nuclear processes such as DNA repair, transcription, and cell cycle, has been studied thoroughly. However, the cytoplasmic response to DNA damage is poorly understood. Here, we demonstrate that DNA damage triggers dramatic reorganization of the Golgi, resulting in its dispersal throughout the cytoplasm. We further show that DNA-damage-induced Golgi dispersal requires GOLPH3/MYO18A/F-actin and the DNA damage protein kinase, DNA-PK. In response to DNA damage, DNA-PK phosphorylates GOLPH3, resulting in increased interaction with MYO18A, which applies a tensile force to the Golgi. Interference with the Golgi DNA damage response by depletion of DNA-PK, GOLPH3, or MYO18A reduces survival after DNA damage, whereas overexpression of GOLPH3, as is observed frequently in human cancers, confers resistance to killing by DNA-damaging agents. Identification of the DNA-damage-induced Golgi response reveals an unexpected pathway through DNA-PK, GOLPH3, and MYO18A that regulates cell survival following DNA damage.

GOLPH3L antagonizes GOLPH3 to determine Golgi morphology.

GOLPH3 is a phosphatidylinositol-4-phosphate (PI4P) effector that plays an important role in maintaining Golgi architecture and anterograde trafficking. GOLPH3 does so through its ability to link trans-Golgi membranes to F-actin via its interaction with myosin 18A (MYO18A). GOLPH3 also is known to be an oncogene commonly amplified in human cancers. GOLPH3L is a GOLPH3 paralogue found in all vertebrate genomes, although previously it was largely uncharacterized. Here we demonstrate that although GOLPH3 is ubiquitously expressed in mammalian cells, GOLPH3L is present in only a subset of tissues and cell types, particularly secretory tissues. We show that, like GOLPH3, GOLPH3L binds to PI4P, localizes to the Golgi as a consequence of its PI4P binding, and is required for efficient anterograde trafficking. Surprisingly, however, we find that perturbations of GOLPH3L expression produce effects on Golgi morphology that are opposite to those of GOLPH3 and MYO18A. GOLPH3L differs critically from GOLPH3 in that it is largely unable to bind to MYO18A. Our data demonstrate that despite their similarities, unexpectedly, GOLPH3L antagonizes GOLPH3/MYO18A at the Golgi.

The PH domain proteins IPIP27A and B link OCRL1 to receptor recycling in the endocytic pathway.

Mutation of the inositol polyphosphate 5-phosphatase OCRL1 results in two disorders in humans, namely Lowe syndrome (characterized by ocular, nervous system, and renal defects) and type 2 Dent disease (in which only the renal symptoms are evident). The disease mechanisms of these syndromes are poorly understood. Here we identify two novel OCRL1-binding proteins, termed inositol polyphosphate phosphatase interacting protein of 27 kDa (IPIP27)A and B (also known as Ses1 and 2), that also bind the related 5-phosphatase Inpp5b. The IPIPs bind to the C-terminal region of these phosphatases via a conserved motif similar to that found in the signaling protein APPL1. IPIP27A and B, which form homo- and heterodimers, localize to early and recycling endosomes and the trans-Golgi network (TGN). The IPIPs are required for receptor recycling from endosomes, both to the TGN and to the plasma membrane. Our results identify IPIP27A and B as key players in endocytic trafficking and strongly suggest that defects in this process are responsible for the pathology of Lowe syndrome and Dent disease.

Differential clathrin binding and subcellular localization of OCRL1 splice isoforms.

Mutation of the inositol polyphosphate 5-phosphatase OCRL1 causes the X-linked disorder oculocerebrorenal syndrome of Lowe, characterized by defects in the brain, kidneys, and eyes. OCRL1 exists as two splice isoforms that differ by a single exon encoding 8 amino acids. The longer protein, termed isoform a, is the only form in brain, whereas both isoforms are present in all other tissues. The significance of OCRL1 splicing is currently unclear. Given its proximity to a clathrin-binding site, we hypothesized that splicing may alter the clathrin binding properties of OCRL1. Here we show that this is indeed the case. OCRL1 isoform a binds clathrin with higher affinity than isoform b and is significantly more enriched in clathrin-coated trafficking intermediates. We also identify a second clathrin-binding site in OCRL1 that contributes to clathrin binding of both isoforms. Association of OCRL1 with clathrin-coated intermediates requires membrane association through interaction with Rab GTPases but not binding to the clathrin adaptor AP2. Expression of OCRL1 isoform a lacking the 5-phosphatase domain impairs transferrin endocytosis, whereas an equivalent version of isoform b does not. Our results suggest that OCRL1 exists as two functional pools, one participating in clathrin-mediated trafficking events such as endocytosis and another that is much less or not involved in this process.