Structure of the gamma heavy chain of the outer arm dynein from Chlamydomonas flagella.
We describe here the vanadate-dependent photocleavage of the gamma heavy chain from the Chlamydomonas outer arm dynein and the pathways by which this molecule is degraded by endoproteases. UV irradiation in the presence of ATP, Mg2+, and vanadate cleaves the gamma chain at a single site (termed V1) to yield fragments of Mr 235,000 and 180,000. Irradiation in the presence of vanadate and Mn2+ results in cleavage of the gamma chain at two other sites (termed V2a and V2b) to yield fragment pairs of Mr 215,000/200,000 and 250,000/165,000. The mass of the intact chain is therefore estimated to be 415,000 D. We have located the major tryptic and staphylococcal protease cleavage sites in the gamma chain, determined the origins of the resulting fragments, and identified the regions which contain the epitopes recognized by two different monoclonal antibodies. Both antibodies react with the smaller V1 fragment; the epitope recognized by antibody 25-8 is within 9,000-52,000 D of the original gamma-chain terminus contained in that fragment, whereas that recognized by antibody 12 gamma B is within 16,000 D of the V1 site. The data permit the construction of a linear map showing the structural organization of the polypeptide. The substructure of the gamma chain is similar to that of the alpha and beta chains of the outer arm dynein with regard to polarity as defined by the sites of vanadate-dependent photocleavage, and to that of the beta chain with regard to a highly sensitive protease site located approximately 10,000 D from the original terminus contained in the smaller V1 fragment.
King
S M
SM
Cell Biology Group, Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545.
Witman
G B
GB
D000251:Adenosine Triphosphatases / Q000276:immunology; D000911:Antibodies, Monoclonal; D002696:Chlamydomonas / Q000166:cytology; D004398:Dyneins / Q000276:immunology; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes; D005407:Flagella / Q000166:cytology; D010447:Peptide Hydrolases / Q000494:pharmacology; D010449:Peptide Mapping; D010782:Photolysis; D012697:Serine Endopeptidases / Q000494:pharmacology; D014638:Vanadates / Q000502:physiology
D000911:Antibodies, Monoclonal; D000939:Epitopes; D014638:Vanadates; D010447:Peptide Hydrolases; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D000251:Adenosine Triphosphatases; D004398:Dyneins