Control of vesicle polarisation during bud growth in Saccharomyces cerevisiae: Role of Rab/Ypt proteins and light chain myosin in controlling the vectorial transport of vesicles

Research project P 14272 Vectoial transport of vesicles in Saccharomyces cerevisiae Antonella RAGNINI- WILSON 06.03.2000 In all eukaryotes asymmetric growth is required to create specialised domains critical for the function of particular cell types or to mediate specific interactions during cell development. Nutrient absorption, neural growth and nerve impulse transmission, development of fertilized eggs into a multicellular organism, budding and mating in yeast cells, are just some examples of processes that require cell polarity. Vectorial transport of vesicles is a key feature of polarissed growth which is coordinated with the organisation of the actin cytoskeleton and the progression of the cell cycle. Its deregulation has been shown to lead to several genetic human diseases and malignant cell growth. Understanding vectorial transport and its coordiantion with other processes in polarized growth will be important in identifying targets for pharmacological research. Yeast provides an excellent eukaryotic model for such studies because much of the machinery for cell morphogenesis is conserved from yeast to man. In the budding yeast Saccharomyces cerevisiae, polarised transport is necessary during bud development and mating. Both processes require rapid plasma membrane growth and cell wall remodeling involving the transport of proteins and enzymes to specific sites on the cell surface. The actin cytoskeleton has an important role in orienting transport vesicles to the budding site. How actin polarisation and vesicle movement are coordinated in time and space is poorly understood. Likewise, many of the molecular players required for the anchoring and movement of exocytic vesicles along actin microfilaments during polarised growth have not yet been identified. The unconventional class V myosin Myo2p is the only known candidate for a motor protein involved in vectorial vesicle transport but how Myo2p interacts with its cargo and how its activity is regulated is unknown. Light chain myosins, kinesin-like molecules, Rab and Rho-like small G proteins are likely to be involved, but direct evidence is missing. The aim of this project is to clarify the role of Rab proteins and light chain myosin in promoting vesicle movement and to identify additional factors involved in this process.

At the start of this project it was not known how Rab/Ypt proteins could act in controlling vesicle and organelle movement in eukaryotes . In this project we have addressed the question of how Rab/Ypt small GTPases could interact with the vesicle/motor anchoring machinery using as tools the powerful genetic system of Saccharomyces cerevisiae. We were able to identify the components of the complex that allows vesicle anchoring to the motor protein that guides vesicles to the final destination during polarised growth, namely the Rab/Ypt protein Sec4p and the class V myosin Myo2. In addition by study the function of one of the proteins associated to this complex, namely the Mlc1p, we show that vesicle delivery to the center of the septum and thus cytokinesis depends on the functional interaction of Mlc1p with the IQ motifs of Myo2p.