Abstract
AbstractWe used genetically-encoded fluorescent probes to visualize the distribution of phosphatidylserine (PS) in live S. cerevisiae. The majority of the PS was found to reside in the cytosolic leaflet of the plasma membrane. Remarkably, PS was polarized, accumulating in bud necks, the bud cortex and the tips of mating projections. Polarization required vectorial delivery of PS-enriched secretory and recycling vesicles. Rapid dissipation of the PS gradient is prevented by the slow diffusion of lipids along the plasmalemmal inner leaflet, estimated by photobleaching recovery measurements to be over an order of magnitude slower than in mammalian cells. In mutants lacking PS-synthase the absence of PS was associated with, and likely responsible for impaired polarization of the Cdc42 complex, leading to inhibition of bud emergence, diminished growth rate and abolishment of mating. The results indicate that PS polarization is required for optimal Cdc42 targeting and activation during cell division and mating.
Highlights
The establishment of polarity is key to cellular replication and to the development and function of multicellular organisms
Progress in understanding PS physiology has been hampered by our inability to visualize this phospholipid inside live cells. This limitation was recently overcome by the development of fluorescent PS biosensors based on the ability of the discoidin C2 domain to selectively recognize PS 14. Using one such genetically-encoded biosensor we show here that PS accumulates preferentially in the plasma membrane of S. cerevisiae but, surprisingly, that it is unevenly distributed within the plasma membrane
As found in mammalian cells 14, the PS probe accumulated in the plasma membrane of yeast with no detectable signal in the endoplasmic reticulum or mitochondria, which are sites of PS synthesis and decarboxylation, respectively
Summary
The establishment of polarity is key to cellular replication and to the development and function of multicellular organisms. Because they are genetically tractable, yeast have become a useful model for the study of cell polarity. Cdc[42] along with Cdc[24] are enriched at the incipient bud site and at the tip of the mating projections that form in response to pheromones 2,3. As for other Rho-family GTPases, the activity of Cdc[42] is dictated by its degree of GTP-occupancy, and stimulation of nucleotide exchange initiates polarization of the actin cytoskeleton at presumptive bud sites or in response to mating pheromones 4. Several factors, including actin cables and the adaptor protein Bem[1], appear to influence or stabilize these loops by aiding in the delivery and retention of Cdc[42] and its effectors 3,5-7
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