Previous and current research
The growth, polarization and cell-to-cell communication of eukaryotic cells requires transport of newly synthesized proteins to the plasma membrane. Intracellular transport is mediated by vesicular carriers and relies on a highly regulated sequence of steps, each of which consists of budding, targeting and fusion of vesicles. We are interested in the general mechanisms controlling membrane transport and their coordination with other cellular functions, e.g. signaling.

Our model system to analyze the molecular basis of intracellular transport is the budding yeast Saccharomyces cerevisiae. The advantage of this model over more complex eukaryotic systems is its easy accessibility to genetic analysis and manipulation. Our lab is particularly interested in Golgi-to-plasma membrane transport. The process of sorting of proteins at the exit from the Golgi, the trans Golgi network (TGN), into different routes to the cell surface is poorly understood on a molecular level. Also, coats for exocytic vesicles have until recently been completely unknown. The key issue of our research is therefore to identify factors involved in these processes using yeast genetics and apply genetic, molecular and cell biological analysis to study their function. We are approaching this goal from three different angles:

First, we study the role of phosphoinositide (PI) lipids in the formation of post-Golgi carriers. PI’s play a critical role in the spatial and temporal regulation of trafficking. We and others have previously shown that phosphatidylinositol 4-phosphate (PI(4)P) is required for exocytosis, TGN-to-vacuole transport and Golgi homeostasis. We have now used genetic screening approaches to identify regulators and targets of PI(4)P signaling in these processes. We discovered that monomeric clathrin adaptors, the GGA proteins, require PI(4)P and the activated small GTPase Arf1p for their recruitment to the TGN and cargo transport to the vacuole as well as to the cell surface. Other candidate regulators of PI(4)P signaling that reveal a tight link to early endosome-TGN transport are currently under investigation.

Second, we investigate the mechanisms of regulation of acute PI levels on membranes. The synthesis and turnover of PI’s is temporally and spatially regulated through activation and highly specific localization of well-conserved lipid kinases and phosphatases that modify the phosphorylation status of the inositol headgroup. Little is known about the regulation of these enzymes and the coordination of PI signaling on membranes with other cellular processes. We have recently discovered a novel mechanism of nucleo-cytoplasmic shuttling of the Golgi PI 4-kinase Pik1p that is regulated by 14-3-3 proteins. This shuttling occurs under certain nutrient conditions and might serve to coordinate Golgi trafficking with nutrient signaling and growth. We aim at understanding the molecular basis of this regulation, the signaling pathways involved and the nuclear roles of Pik1p and its product PI(4)P.

Since PI’s often function together with small GTPases (e.g. Arf1p) in the recruitment of cytosolic effectors, a process called coincidence detection, we are currently studying how the generation of these dual-key signals is regulated. This work includes investigation of the crosstalk of Arf- and Rab GTPases, and their relationship to PI signaling.

Third, we are using genetic approaches to isolate the still mostly unknown machinery for sorting at the TGN into specialized routes to the cell surface. In a joint project with Kai Simon’s group, we have isolated new candidate molecules for a role in sorting that we now want to study in detail. In an ongoing visual screen looking directly at cargo behavior during the process of exocytosis, we are particularly interested in finding factors that are involved in sorting decisions for nutrient-regulated permeases. In this project, our long-term goal is a comprehensive analysis of TGN-to-plasma membrane transport in a combination of high-throughput screening with cell biological and biochemical analysis.