The Roadblock light chains are ubiquitous components of cytoplasmic dynein that form homo- and heterodimers.
The Roadblock/LC7 class of light chains associate with the intermediate chains at the base of the soluble dynein particle. In mammals, there are two Roadblock isoforms (Robl1 and Robl2), one of which (Robl2) is differentially expressed in a tissue-dependent manner and is especially prominent in testis. Here we define the alpha helical content of Robl and demonstrate using both the yeast two-hybrid system and in vitro biochemistry that Robl1 and Robl2 are capable of forming homo- and heterodimers. This is the first report of heterodimer formation by any cytoplasmic dynein component, and it further enlarges the number of potential cytoplasmic dynein isoforms available for binding specific cellular cargoes. In addition, we have generated an antibody that specifically recognizes Robl light chains and shows a 5-10 fold preference for Robl2 over Robl1. Using this antibody, we show that Robl is a ubiquitous cytoplasmic dynein component, being found in samples purified from brain, liver, kidney, and testis. Immunofluorescence analysis reveals that Robl is present in punctate organelles in rat neuroblastoma cells. In testis, Robl is found in Leydig cells, spermatocytes, and sperm flagella.
Nikulina
Karina
K
Department of Biochemistry, University of Connecticut Health Center, Farmington 06030-3305, USA.
Patel-King
Ramila S
RS
Takebe
Sachiko
S
Pfister
K Kevin
KK
King
Stephen M
SM
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity / Q000276:immunology; D015153:Blotting, Western; D001923:Brain Chemistry; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D002499:Centrifugation, Density Gradient; D002942:Circular Dichroism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004398:Dyneins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D017930:Genes, Reporter / Q000235:genetics; D007150:Immunohistochemistry; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007668:Kidney / Q000737:chemistry; D007985:Leydig Cells / Q000737:chemistry; D008099:Liver / Q000737:chemistry; D008297:Male; D058619:Maltose-Binding Proteins; D051379:Mice; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000737:chemistry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D017433:Protein Structure, Secondary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000737:chemistry / Q000276:immunology; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013094:Spermatozoa / Q000737:chemistry; D013737:Testis / Q000737:chemistry; D020798:Two-Hybrid System Techniques; D014611:Vaccination
D002352:Carrier Proteins; D018076:DNA, Complementary; D007527:Isoenzymes; D058619:Maltose-Binding Proteins; D011993:Recombinant Fusion Proteins; D004398:Dyneins