Regulation of lipid signaling at the Golgi by the lipid phosphatases hSAC1 and OCRL1

Phosphoinositides are key lipid signaling molecules present in membranes of all eukaryotes. Different species of phosphoinositides serve as membrane signposts at distinct cellular compartments. This assymetric distribution of phosphoinositides is achieved by the presence of an elaborate set of lipid kinases and phosphatases operating at specific organelle membranes. The lipid phosphatase SAC1 is found in both endoplasmic reticulum (ER) and Golgi. Similar to yeast Sac1p, human SAC1 (hSAC1) is the major phosphatidylinositol-4-phosphatase (PI(4)P-phosphatase). Distinct localization of hSAC1 in both ER and Golgi membranes suggests that this PI(4)P-phosphatase has compartment specific roles in regulating steady state distribution of PI(4)P in these organelles. OCRL1 is a Golgi and endosomal localized PI(4,5)P2 5-phosphatase that is implicated in a severe X-linked disease, Lowe syndrome, which is characterized by congenital cataracts, Fanconi syndrome and mental retardation. How mutations in OCRL1 cause Lowe syndrome is unknown. The functional analysis of hSAC1 and OCRL1 in regulating Golgi PI(4)P and PI(4,5)P2 is the main focus of this work. Confocal immunofluorescence and immuno-electron microscopy (immuno-EM) show that PI(4)P and hSAC1 form an opposing gradient in the Golgi. hSAC1 is highly enriched at Golgi cisternal membranes while PI(4)P is concentrated at the trans-Golgi network (TGN) where cargo proteins are packaged and exported. Golgi enzymes such as N-acetylglucosaminyltransferase I (NacT1) and mannosidase II (ManII) are preferentially found in these PI(4)P-depleted areas. siRNA-mediated knock-down of hSAC1 leads to accumulation of PI(4)P at Golgi, plasma membrane and endosomal like structures and causes mislocalization of ManII and NacT1. This data suggests that SAC1 establishes PI(4)P-depleted Golgi regions that are important for proper localization and recycling of Golgi resident enzymes. Conversely, depletion of OCRL1 does not disturb Golgi morphology or induce mislocalization of Golgi resident enzymes. However, bulk secretion is inhibited in OCRL1 depleted cells. The OCRL1-b splice variant populates TGN and early endosomal compartment whereas the OCRL1-a splice variant containing an extra 8 amino acid acidic cluster is found only in a subset of late endosomal/ lysosomal membranes. This distinct localization of OCRL1 splice variants indicates that each isoform might regulate different trafficking routes by regulating PI(4,5)P2 levels at these compartments. Together, the results show that hSAC1 and OCRL1 establish distinct phosphoinositide-specific domains within the Golgi that are instrumental for segregation of anterograde trafficking from the recycling of resident Golgi enzymes.

Level thesis:degree_level

thesis.doctoral

Grantor dc:publisher

Universität Heidelberg

Year

2007

Author dc:creator

• Cheong, Fei Ying

Contributors dc:contributor

• Mayinger, Peter PD

Repository record source_url

http://www.ub.uni-heidelberg.de/archiv/7101

OAI identifier oai:identifier

oai:archiv.ub.uni-heidelberg.de:7101