Phosphorylated derivatives of phosphatidylinositiol, also called phosphoinositides, orchestrate a multitude of cellular processes via downstream lipid-binding effector proteins. The temporal and spatial control of phosphoinositides is critical for their regulatory function. These lipid-signaling molecules are particularly important for coordination of distinct membrane trafficking reactions. The fact that mutations in several lipid phosphatases are linked to a variety of serious diseases such as kidney disease, diabetes, severe congenital disorders and cancer, indicates that these enzymes have vital cellular functions. Currently, we focus on a detailed characterization of two specific lipid phosphatases in yeast and in mammalian cells.

Characterization of Sac1 lipid phosphatases

Our recent work shows that the yeast Sac1 lipid phosphatase controls separate pools of cellular phosphoinositides. Sac1p plays a pivotal role in coordinating endoplasmic reticulum (ER) and Golgi function in response to nutrients and cell growth rates. In yeast, we have found that dolicholphosphate mannose synthase Dpm1p, an essential ER enzyme involved in glycosylation, recruits Sac1p to ER membranes during times of rapid cell division. Nutrient limitation slows cell proliferation and triggers dissociation of Sac1p from Dpm1p, causing accumulation of this lipid phosphatase at the Golgi. The cell growth-dependent shuttling of Sac1p between ER and Golgi is responsible for control of ER and Golgi function in yeast. Synchronizing the secretory pathway with cell proliferation by a lipid phosphatase may therefore constitute a novel and general mechanism to regulate intracellular membrane trafficking and homeostasis

The mammalian versions of Sac1 are functionally conserved and populate the same compartments as the yeast Sac1 phosphatase. In contrast to the yeast Sac1 protein, all mammalian Sac1 orthologs have a canonical KXKXX ER localization motif at their C termini, indicating that ER localization of these proteins is mediated by their recruitment into retrograde transport vesicles at the Golgi. In immunoprecipitation experiments, we were able to identify members of the coatomer I (COPI) complex as interaction partners of SAC1. We found that mutation of the putative C terminal COPI-interaction motif (KEKID) abolishes interaction with COPI and causes accumulation of SAC1 in the Golgi.

We are presently characterizing mechanism and regulation of cell growth-dependent relocation of yeast Sac1 between ER and Golgi. We also analyze how Sac1 regulates oligosaccharide bioysynthesis and glycosylation at the ER as well as ER/Golgi homeostasis.

Lipid phosphatase activity in the Golgi and its implications in Lowe syndrome

In the past few years, an increasing number of disease-related mutations have been mapped to genes encoding lipid phosphatases. With a few exceptions the molecular mechanisms for developing clinical symptoms upon disturbance of phosphoinositide metabolism remain unknown. The oculocerebrorenal syndrome of Lowe (OCRL) is a particular prominent example of a multisystem disorder that is based on mutations in a phosphatidylinositol 5-phosphatase. Although the exact cause for development of OCRL is not understood, the localization of the OCRL1 lipid phosphatase to Golgi membranes indicated that this syndrome is based on defects in lipid signaling at this organelle. In addition to OCRL1, Golgi phosphoinositides are also controlled by the polyphosphoinositide phosphatase SAC1. Whether mutations in the SAC1 gene cause human diseases is unknown. A major goal of our lab is the cell biological characterization of phosphoinositide signaling at the Golgi. The two phosphoinositides PtdIns(4)P and PtdIns(4,5)P2 have both been implicated in Golgi structural integrity and trafficking. Our preliminary data indicate that these lipids populate separate regions within the Golgi. While the kinases that synthesize these lipids have been characterized, the role of lipid phosphatases in phosphoinositide signaling at the Golgi remains unclear. We are elucidating how OCRL1 regulates PtdIns(4,5)P2 levels at Golgi membranes. We are also studying how OCRL function contributes to controlling specific membrane trafficking reactions. This analysis should lay the ground work to eventually understand the development of the specific sets of clinical manifestations seen in Lowe syndrome patients.