title: Coordinate action of distinct sequence elements localizes checkpoint kinase Hsl1 to the septin collar at the bud neck in Saccharomyces cerevisiae.
abstract: Passage through the eukaryotic cell cycle requires processes that are tightly regulated both spatially and temporally. Surveillance mechanisms (checkpoints) exert quality control and impose order on the timing and organization of downstream events by impeding cell cycle progression until the necessary components are available and undamaged and have acted in the proper sequence. In budding yeast, a checkpoint exists that does not allow timely execution of the G2/M transition unless and until a collar of septin filaments has properly assembled at the bud neck, which is the site where subsequent cytokinesis will occur. An essential component of this checkpoint is the large (1518-residue) protein kinase Hsl1, which localizes to the bud neck only if the septin collar has been correctly formed. Hsl1 reportedly interacts with particular septins; however, the precise molecular determinants in Hsl1 responsible for its recruitment to this cellular location during G2 have not been elucidated. We performed a comprehensive mutational dissection and accompanying image analysis to identify the sequence elements within Hsl1 responsible for its localization to the septins at the bud neck. Unexpectedly, we found that this targeting is multipartite. A segment of the central region of Hsl1 (residues 611-950), composed of two tandem, semiredundant but distinct septin-associating elements, is necessary and sufficient for binding to septin filaments both in vitro and in vivo. However, in addition to 611-950, efficient localization of Hsl1 to the septin collar in the cell obligatorily requires generalized targeting to the cytosolic face of the plasma membrane, a function normally provided by the C-terminal phosphatidylserine-binding KA1 domain (residues 1379-1518) in Hsl1 but that can be replaced by other, heterologous phosphatidylserine-binding sequences.
authors.affiliation: Division of Biochemistry, Biophysics and Structural Biology and Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720.
authors.forename: Gregory C
authors.initials: GC
authors.lastname: Finnigan
authors.affiliation: Division of Biochemistry, Biophysics and Structural Biology and Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720.
authors.forename: Sarah M
authors.initials: SM
authors.lastname: Sterling
authors.affiliation: Division of Biochemistry, Biophysics and Structural Biology and Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720.
authors.forename: Angela
authors.initials: A
authors.lastname: Duvalyan
authors.affiliation: Division of Biochemistry, Biophysics and Structural Biology and Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720.
authors.forename: Elizabeth N
authors.initials: EN
authors.lastname: Liao
authors.affiliation: Division of Biochemistry, Biophysics and Structural Biology and Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720.
authors.forename: Aspram
authors.initials: A
authors.lastname: Sargsyan
authors.affiliation: Division of Biochemistry, Biophysics and Structural Biology and Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720.
authors.forename: Galo
authors.initials: G
authors.lastname: Garcia
authors.affiliation: Division of Biochemistry, Biophysics and Structural Biology and Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720 Life Sciences Division, Lawrence Berkeley National Laboratory, and Howard Hughes Medical Institute, Berkeley, CA 94720.
authors.forename: Eva
authors.initials: E
authors.lastname: Nogales
authors.affiliation: Division of Biochemistry, Biophysics and Structural Biology and Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720 jthorner@berkeley.edu.
authors.forename: Jeremy
authors.initials: J
authors.lastname: Thorner
mesh_terms: D000595:Amino Acid Sequence; D018797:Cell Cycle Proteins / Q000378:metabolism; D002455:Cell Division / Q000502:physiology; D003599:Cytoskeleton / Q000378:metabolism; D059565:G2 Phase Cell Cycle Checkpoints; D018816:Genes, cdc; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism*; D004718:Saccharomycetales / Q000235:genetics / Q000378:metabolism; D058112:Septins / Q000235:genetics / Q000378:metabolism*
chemical_list: D018797:Cell Cycle Proteins; D029701:Saccharomyces cerevisiae Proteins; C106494:HSL1 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; D058112:Septins