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Volume 4, Issue 3

Characterization of Bud3 domains sufficient for bud neck targeting in Open Access

Abstract

The cytoskeleton serves a diverse set of functions in both multi- and unicellular organisms, including movement, transport, morphology, cell division and cell signalling. The septin family of cytoskeletal proteins are found within all fungi and metazoans and can generate three-dimensional scaffolds that promote membrane curvature, serve as physical barriers and coordinate cell cycle checkpoints. In budding yeast, the septins organize into polymerized filaments that decorate the division site between mother and daughter cells during mitosis; assembly of this structure at the ‘bud neck’ is critical for completion of cytokinesis and execution of numerous other cellular events. One such pathway includes bud site selection and the recruitment of proteins such as Bud4 and Bud3 that are responsible for promoting an axial budding pattern in haploid yeast. While Bud4 appears to be recruited to the septins independently of the presence of Bud3, it is likely that Bud3 can localize to the bud neck using both Bud4-dependent and Bud4-independent mechanisms. Furthermore, it remains unclear which precise domain or domains within Bud3 is/are both necessary and sufficient for optimal association at the septin structure. In this study, we examined the localization of GFP-Bud3 constructs in otherwise wild-type (WT) haploid yeast cells expressing Cdc10-mCherry using fluorescence microscopy; we tested a collection of N- and C-terminal truncations and fusions of separate Bud3 protein elements to identify the smallest domain(s) responsible for bud neck localization. We found that the coordinate action of the central amphipathic helix (residues 847–865) and a partially conserved C-terminal motif (residues 1172–1273) was sufficient to promote bud neck recruitment in the presence of endogenous Bud3. This domain is considerably smaller than the previously characterized C-terminal portion required to physically interact with Bud4 (1221–1636) and utilizes a similar mechanism of pairing membrane association, with a separate localization domain, similar to other non-septin proteins targeted to the division site during cell division.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): bud neck , Bud3 , fluorescence microscopy and septins
Funding
This study was supported by the:
  • National Institute of Food and Agriculture (Award Hatch Project 1013520)
    • Principle Award Recipient: GregoryC Finnigan
  • National Institute of General Medical Sciences (Award P20 GM103418)
    • Principle Award Recipient: GregoryC Finnigan
© 2022 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License.
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2022-03-24
2024-04-24
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Characterization of Bud3 domains sufficient for bud neck targeting in S. cerevisiae
Access Microbiology 4, 000341 (2022); https://doi.org/10.1099/acmi.0.000341
/content/journal/acmi/10.1099/acmi.0.000341
/content/journal/acmi/10.1099/acmi.0.000341
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