Identification of a molecular chaperone in the eukaryotic flagellum and its localization to the site of microtubule assembly.
Monoclonal antibodies that recognize HSP70 family members from evolutionarily divergent organisms were used to identify both constitutively-expressed and stress-inducible HSP70 proteins in the green alga Chlamydomonas. These monoclonal antibodies also cross-reacted with a 70 kDa flagellar protein that comigrated with the constitutively-expressed HSP70 isoform(s) present in the cell body; this is the first identification of a molecular chaperone within the eukaryotic flagellum. Fractionation experiments demonstrated that much of the flagellar HSP70 was bound to the '9+2' microtubule axoneme. Incubation of isolated axonemes in ATP, but not AMP or AMP-PNP, caused significant release of the previously bound HSP70 as is characteristic of complexed HSP70s. Immunofluorescent localization in whole flagella showed that flagellar HSP70 was concentrated at the distal ends of flagella, sites of axonemal assembly in vivo. Extraction of axonemes under ionic conditions known to cause the release of capping structures that link the distal ends of the axonemal microtubules to the flagellar membrane also caused the release of axonemal-bound HSP70. Taken together, these results suggest a model in which an HSP70 chaperone may assist in targeting tubulin and other unassembled axonemal components to the flagellar tip where the chaperone may also participate in the assembly of the '9+2' flagellar axoneme.
Bloch
M A
MA
Department of Molecular, Cellular and Developmental Biology, Haverford College, PA 19041, USA.
Johnson
K A
KA
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D000911:Antibodies, Monoclonal; D002696:Chlamydomonas / Q000737:chemistry* / Q000648:ultrastructure; D003429:Cross Reactions; D000939:Epitopes / Q000032:analysis; D005407:Flagella / Q000737:chemistry*; D005455:Fluorescent Antibody Technique; D018840:HSP70 Heat-Shock Proteins / Q000032:analysis / Q000276:immunology; D015151:Immunoblotting; D008870:Microtubules / Q000737:chemistry* / Q000378:metabolism; D018832:Molecular Chaperones / Q000032:analysis*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D018840:HSP70 Heat-Shock Proteins; D018832:Molecular Chaperones; D000255:Adenosine Triphosphate