title: Application of a new dual localization-affinity purification tag reveals novel aspects of protein kinase biology in Aspergillus nidulans.
abstract: Filamentous fungi occupy critical environmental niches and have numerous beneficial industrial applications but devastating effects as pathogens and agents of food spoilage. As regulators of essentially all biological processes protein kinases have been intensively studied but how they regulate the often unique biology of filamentous fungi is not completely understood. Significant understanding of filamentous fungal biology has come from the study of the model organism Aspergillus nidulans using a combination of molecular genetics, biochemistry, cell biology and genomic approaches. Here we describe dual localization-affinity purification (DLAP) tags enabling endogenous N or C-terminal protein tagging for localization and biochemical studies in A. nidulans. To establish DLAP tag utility we endogenously tagged 17 protein kinases for analysis by live cell imaging and affinity purification. Proteomic analysis of purifications by mass spectrometry confirmed association of the CotA and NimXCdk1 kinases with known binding partners and verified a predicted interaction of the SldABub1/R1 spindle assembly checkpoint kinase with SldBBub3. We demonstrate that the single TOR kinase of A. nidulans locates to vacuoles and vesicles, suggesting that the function of endomembranes as major TOR cellular hubs is conserved in filamentous fungi. Comparative analysis revealed 7 kinases with mitotic specific locations including An-Cdc7 which unexpectedly located to mitotic spindle pole bodies (SPBs), the first such localization described for this family of DNA replication kinases. We show that the SepH septation kinase locates to SPBs specifically in the basal region of apical cells in a biphasic manner during mitosis and again during septation. This results in gradients of SepH between G1 SPBs which shift along hyphae as each septum forms. We propose that SepH regulates the septation initiation network (SIN) specifically at SPBs in the basal region of G1 cells and that localized gradients of SIN activity promote asymmetric septation.
authors.affiliation: Department of Molecular Genetics, The Ohio State University, Columbus, Ohio, United States of America.
authors.forename: Colin P
authors.initials: CP
authors.lastname: De Souza
authors.affiliation: Department of Molecular Genetics, The Ohio State University, Columbus, Ohio, United States of America.
authors.forename: Shahr B
authors.initials: SB
authors.lastname: Hashmi
authors.affiliation: Department of Molecular Genetics, The Ohio State University, Columbus, Ohio, United States of America.
authors.forename: Aysha H
authors.initials: AH
authors.lastname: Osmani
authors.affiliation: Department of Molecular Genetics, The Ohio State University, Columbus, Ohio, United States of America.
authors.forename: Stephen A
authors.initials: SA
authors.lastname: Osmani
mesh_terms: D000595:Amino Acid Sequence; D001233:Aspergillus nidulans / Q000166:cytology / Q000187:drug effects / Q000201:enzymology* / Q000254:growth & development; D001542:Benomyl / Q000494:pharmacology; D002467:Cell Nucleus / Q000187:drug effects / Q000201:enzymology; D002846:Chromatography, Affinity / Q000379:methods*; D022162:Cytoplasmic Vesicles / Q000187:drug effects / Q000201:enzymology; D005656:Fungal Proteins / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007399:Interphase / Q000187:drug effects; D018386:Kinetochores / Q000187:drug effects / Q000201:enzymology; D008870:Microtubules / Q000187:drug effects / Q000201:enzymology; D008938:Mitosis / Q000187:drug effects; D008969:Molecular Sequence Data; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects; D040901:Proteomics; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D064048:Spindle Pole Bodies / Q000187:drug effects / Q000201:enzymology; D014617:Vacuoles / Q000187:drug effects / Q000201:enzymology
chemical_list: D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D011494:Protein Kinases; D001542:Benomyl