Molecular requirements for the inter-subunit interaction and kinetochore recruitment of SKAP and Astrin.
Accurate chromosome segregation during cell division is crucial for propagating life and protects from cellular transformation. The SKAP:Astrin heterodimer localizes to spindle microtubules and to mature microtubule-kinetochore attachments during mitosis. Depletion of either subunit disrupts spindle structure and destabilizes kinetochore-microtubule attachments. Here, we identify molecular requirements for the inter-subunit interaction of SKAP and Astrin, and discuss requirements for their kinetochore recruitment. We also identify and characterize a microtubule-binding domain in SKAP, distinct from the SXIP motif that mediates end binding (EB) protein binding and plus end tracking, and show that it stimulates the growth-rate of microtubules, possibly through a direct interaction with tubulin. Mutations targeting this microtubule-binding domain impair microtubule plus-end tracking but not kinetochore targeting, and recapitulate many effects observed during depletion of SKAP. Collectively, our studies represent the first thorough mechanistic analysis of SKAP and Astrin, and significantly advance our functional understanding of these important mitotic proteins.
Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Straße 11, 44227 Dortmund, Germany.
Alexandra
A
Friese
Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Straße 11, 44227 Dortmund, Germany.
Alex C
AC
Faesen
Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Straße 11, 44227 Dortmund, Germany.
Pim J
PJ
Huis in 't Veld
Gene Center Munich, Ludwig-Maximilians-Universität München, Feodor-Lynen-Straße 25, 81377 Munich, Germany.
Josef
J
Fischböck
Department of Structural Biochemistry, Max Planck Institute of Molecular Physiology, Otto-Hahn-Straße 11, 44227 Dortmund, Germany.
Daniel
D
Prumbaum
Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Straße 11, 44227 Dortmund, Germany.
Arsen
A
Petrovic
Department of Structural Biochemistry, Max Planck Institute of Molecular Physiology, Otto-Hahn-Straße 11, 44227 Dortmund, Germany.
Stefan
S
Raunser
Gene Center Munich, Ludwig-Maximilians-Universität München, Feodor-Lynen-Straße 25, 81377 Munich, Germany.
Franz
F
Herzog
Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Straße 11, 44227 Dortmund, Germany.|Centre for Medical Biotechnology, Faculty of Biology, University Duisburg-Essen, Universitätsstrasse, 45141 Essen, Germany.
Andrea
A
Musacchio
D000595:Amino Acid Sequence; D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020090:Chromosome Segregation; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D018386:Kinetochores / Q000378:metabolism* / Q000648:ultrastructure; D008869:Microtubule-Associated Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism* / Q000648:ultrastructure; D008938:Mitosis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D021381:Protein Transport; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D008941:Spindle Apparatus / Q000378:metabolism* / Q000648:ultrastructure; D014404:Tubulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D018797:Cell Cycle Proteins; C555984:KNSTRN protein, human; D008869:Microtubule-Associated Proteins; D011994:Recombinant Proteins; C486663:SPAG5 protein, human; D014404:Tubulin