Abstract
ABSTRACTThe diversity of microtubule functions is dependent on the status of tubulin C-termini. To address the physiological role of the C-terminal aromatic residue of α-tubulin, a tub1-Glu yeast strain expressing an α-tubulin devoid of its C-terminal amino acid was used to perform a genome-wide-lethality screen. The identified synthetic lethal genes suggested links with endocytosis and related processes. In the tub1-Glu strain, the routing of the v-SNARE Snc1 was strongly impaired, with a loss of its polarized distribution in the bud, and Abp1, an actin patch or endocytic marker, developed comet-tail structures. Snc1 trafficking required dynamic microtubules but not dynein and kinesin motors. Interestingly, deletion of the microtubule plus-end-tracking protein Bik1 (a CLIP170 ortholog), which is preferentially recruited to the C-terminal residue of α-tubulin, similarly resulted in Snc1 trafficking defects. Finally, constitutively active Rho1 rescued both Bik1 localization at the microtubule plus-ends in tub1-Glu strain and a correct Snc1 trafficking in a Bik1-dependent manner. Our results provide the first evidence for a role of microtubule plus-ends in membrane cargo trafficking in yeast, through Rho1- and Bik1-dependent mechanisms, and highlight the importance of the C-terminal α-tubulin amino acid in this process.
Highlights
Microtubules are fibrous structures in eukaryotic cells that play a vital role in cell organization and division
As the α-tubulin C-terminal amino acid has been shown to be crucial for the interaction of CLIP170 and the yeast ortholog Bik1 with microtubule plus-ends through their CAP-Gly domain (BadinLarcon et al, 2004; Honnappa et al, 2006; Peris et al, 2006), we investigated a possible role for Bik1 in mediating the effect of the tub1-Glu mutation
Our results argue for a detrimental role for Bik1 in Snc1 trafficking, likely dependent on its localization at microtubule plus-ends the control of the GTPase Rho1
Summary
Microtubules are fibrous structures in eukaryotic cells that play a vital role in cell organization and division. We generated a budding yeast strain solely expressing an α-tubulin devoid of its C-terminal aromatic residues (tub1-Glu strain) to model detyrosinated Glu-tubulin, as re-addition of phenylalanine is not observed in the tub1-Glu mutant cells (Badin-Larcon et al, 2004). Using this strain, we discovered that the CLIP170 ortholog Bik is able to sense the C-terminal α-tubulin aromatic residue at microtubules plus-ends (Badin-Larcon et al, 2004). Structural studies have established that the C-terminal aromatic residue is required for the direct interaction of α-tubulin with CAP-Gly domains and CLIP170 (Honnappa et al, 2006; Mishima et al, 2007)
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