Characterization of structural and functional domains of the anillin-related protein Mid1p that contribute to cytokinesis in fission yeast.
Fission yeast cells depend on the anillin-related protein Mid1p for reliable cytokinesis. Insolubility limits the purification of full-length Mid1p for biophysical analysis, and lack of knowledge about the structural domains of Mid1p limits functional analysis. We addressed these limitations by identifying in a bacterial expression screen of random Mid1p fragments five soluble segments that can be purified and one insoluble segment. Using complementation experiments in Δmid1 cells, we tested the biological functions of these six putative domains that account for full-length Mid1p. The N-terminal domain (residues 1-149) is essential for correct positioning and orientation of septa. The third domain (residues 309-452) allows the construct composed of the first three domains (residues 1-452) to form hydrodynamically well-behaved octamers. Constructs consisting of residues 1-452 or 1-578 carry out most functions of full-length Mid1p, including concentration at the equatorial cortex in nodes that accumulate myosin-II and other contractile ring proteins during mitosis. However, cells depending on these constructs without the insoluble domain (residues 579-797) form equatorially located rings slowly from strands rather than by direct condensation of nodes. We conclude that residues 1-578 assemble node components myosin-II, Rng2p, and Cdc15p, and the insoluble domain facilitates the normal, efficient condensation of nodes into rings.
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8103, USA.
Shambaditya
S
Saha
Thomas D
TD
Pollard
D002455:Cell Division; D016764:Cell Polarity; D003285:Contractile Proteins / Q000737:chemistry* / Q000378:metabolism*; D048749:Cytokinesis; D005816:Genetic Complementation Test; D057446:Hydrodynamics; D008954:Models, Biological; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D012568:Schizosaccharomyces / Q000166:cytology* / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry* / Q000378:metabolism*; D017384:Sequence Deletion; D012995:Solubility; D013329:Structure-Activity Relationship; D014461:Ultracentrifugation
D003285:Contractile Proteins; C489694:Mid1 protein, S pombe; D029702:Schizosaccharomyces pombe Proteins; C095886:anillin