Novel component of the fission yeast memory-based polarity landmark
Johnson, Hayley Louise
Polarised cells have spatial differences in their shape, structure or function. Polarisation is essential for many cellular processes, including directional growth. This occurs at specific sites within the cell determined by polarity cues. Fission yeast are an excellent model to study how polarity cues are generated and maintained. Fission yeast are rod-shaped cells that grow exclusively from their tips, co-ordinated by two independent internal polarity cues, the Tea1-Tea4 system and the Rax1-Rax2 system. The Tea1-Tea4 polarity cue is highly dynamic and is based on the cell’s existing geometry. Tea1 and Tea4 are delivered to the cell tips on the plus ends of microtubules that run along the length of the cell. The Rax1-Rax2 system is a stable cue and is based on sites of previous growth. Rax1 and Rax2 are delivered to the cell tips through the secretory pathway during active growth. Once delivered, Rax1 and Rax2 are stably retained at the cell tips and are able to recruit growth machinery back to a previously growing cell tip in the next cell cycle. By this mechanism cell is able to ‘remember’ a site of previous cell growth and reinitiate growth from this site. In order to correctly function as a polarity landmark for growth at the cell tips, both Rax1 and Rax2 need to localise correctly. In this work I have shown that large C-terminal tags on Rax2 impair its function, despite Rax2 correctly localising to the cell tips. Additionally, I have shown that Rax1 is required for Rax2 trafficking out of the ER, and that the cytoplasmic 26 amino acids of Rax2 are also required for Rax2 trafficking. I have identified interactors of Rax2-His-Tev-Biotin using cross-linking, purification and mass spectrometry. Known polarity proteins Bgs1, Bgs3, Rga1 and Rga3 were found to interact with Rax2. Additionally, a previously uncharacterised protein, Lrx1, interacts with Rax2 and is important for the correct localisation of Rax1-Rax2 to the cell tips, and thus the correct functioning of the Rax1- Rax2 memory-based polarity cue.