Homology of the 74-kD cytoplasmic dynein subunit with a flagellar dynein polypeptide suggests an intracellular targeting function.
In previous work we found cytoplasmic dynein to be a complex of two catalytic heavy chains and at least seven co-purifying polypeptides of unknown function. The most prominent of these is a 74-kD electrophoretic species which can be resolved as two to three bands by SDS-PAGE. We have now selected a series of overlapping rat brain cDNAs encoding the 74-kD species. The deduced sequence of a full-length cDNA predicts a 72,753 D polypeptide which includes the amino acid sequences of nine peptides determined by NH2-terminal microsequencing. PCR performed on first strand rat brain cDNA together with the sequence of a partially matching tryptic peptide indicated the existence of at least three isoforms of the 74-kD cytoplasmic dynein subunit. Comparison with known sequences revealed that the carboxyl-terminal half of the polypeptide is 26.4% identical and 47.7% similar to the product of the Chlamydomonas ODA6 gene, a 70-kD intermediate chain of flagellar outer arm dynein. Immunoblot analysis with a monoclonal antibody to the 74-kD species indicated a widespread tissue distribution, as expected for a cytoplasmic dynein subunit. Nonetheless, the antibody recognized a 67-kD species in ram sperm flagella and pig tracheal cilia, supporting the existence of distinct but related cytoplasmic and axonemal polypeptides in mammals. In view of evidence for a role for the ODA6 gene product in anchoring flagellar dynein to the A subfiber microtubule in the axoneme, we predict an analogous role for the 74-kD polypeptide, perhaps in mediating the interaction of cytoplasmic dynein with membranous organelles and kinetochores.
Paschal
B M
BM
Cell Biology Group, Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545.
Mikami
A
A
Pfister
K K
KK
Vallee
R B
RB
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology; D002923:Cilia / Q000201:enzymology; D003593:Cytoplasm / Q000201:enzymology*; D004247:DNA / Q000235:genetics; D004398:Dyneins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D005407:Flagella / Q000201:enzymology*; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000737:chemistry; D051381:Rats; D011919:Rats, Inbred Strains; D012756:Sheep; D013082:Sperm Tail / Q000201:enzymology; D013737:Testis / Q000201:enzymology
D012333:RNA, Messenger; D004247:DNA; D004398:Dyneins