Flagellar localization of a novel isoform of myosin, myosin XXI, in Leishmania.
Leishmania major genome analysis revealed the presence of putative genes corresponding to two myosins, which have been designated to class IB and a novel class, class XXI, specifically present in kinetoplastids. To characterize these myosin homologs in Leishmania, we have cloned and over-expressed the full-length myosin XXI gene and variable region of myosin IB gene in bacteria, purified the corresponding proteins, and then used the affinity purified anti-sera to analyze the expression and intracellular distribution of these proteins. Whereas myosin XXI was expressed in both the promastigote and amastigote stages, no expression of myosin IB could be detected in any of the two stages of these parasites. Further, myosin XXI expression was more predominant in the promastigote stage where it was preferentially localized in the proximal region of the flagellum. The observed flagellar localization was not dependent on the myosin head region or actin but was exclusively determined by the myosin tail region, as judged by over-expressing GFP conjugates of full-length myosin XXI, its head domain and its tail domain separately in Leishmania. Furthermore, immunofluorescence and immuno-gold electron microscopy analyses revealed that this protein was partly associated with paraflagellar rod proteins but not with tubulins in the flagellar axoneme. Our results, for the first time, report the expression and detailed analysis of cellular localization of a novel class of myosin, myosin XXI in trypanosomatids.
Katta
Santharam S
SS
Division of Molecular and Structural Biology, Central Drug Research Institute, Lucknow, India.
Sahasrabuddhe
Amogh A
AA
Gupta
Chhitar M
CM
D000818:Animals; D019677:Artificial Gene Fusion; D016054:DNA, Protozoan / Q000737:chemistry / Q000235:genetics; D005407:Flagella / Q000737:chemistry*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D018320:Leishmania major / Q000737:chemistry*; D008856:Microscopy, Fluorescence; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D009218:Myosins / Q000032:analysis* / Q000235:genetics*; D020033:Protein Isoforms / Q000032:analysis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA
D016054:DNA, Protozoan; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D009218:Myosins