Cargo binding and regulatory sites in the tail of fungal conventional kinesin.
Here, using a quantitative in vivo assay, we map three regions in the carboxy terminus of conventional kinesin that are involved in cargo association, folding and regulation, respectively. Using C-terminal and internal deletions, point mutations, localization studies, and an engineered 'minimal' kinesin, we identify five heptads of a coiled-coil domain in the kinesin tail that are necessary and sufficient for cargo association. Mutational analysis and in vitro ATPase assays highlight a conserved motif in the globular tail that is involved in regulation of the motor domain; a region preceding this motif participates in folding. Although these sites are spatially and functionally distinct, they probably cooperate during activation of the motor for cargo transport.
Seiler
S
S
Adolf Butenandt Institute, Cell Biology, University of Munich, 80336 Munich, Germany.
Kirchner
J
J
Horn
C
C
Kallipolitou
A
A
Woehlke
G
G
Schliwa
M
M
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001665:Binding Sites; D017124:Conserved Sequence / Q000235:genetics; D005455:Fluorescent Antibody Technique; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005816:Genetic Complementation Test; D016547:Kinesins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D020409:Molecular Motor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009491:Neurospora / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism; D010641:Phenotype; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D005656:Fungal Proteins; D020409:Molecular Motor Proteins; D011993:Recombinant Fusion Proteins; D000251:Adenosine Triphosphatases; D016547:Kinesins