Erythrocyte tropomodulin isoforms with and without the N-terminal actin-binding domain.
Erythrocyte tropomodulin (E-Tmod or Tmod1) of 41 kDa is a tropomyosin (TM)-binding protein that caps the slow-growing end of the actin filaments. Its N-terminal half is flexible, whereas the C-terminal half has a single domain structure. E-Tmod/TM5 complex may function as a "molecular ruler" generating actin protofilaments of ∼37 nm. Here we report the discovery of a short isoform of 29 kDa that lacks the N-terminal actin-binding domain (N-ABD) but retains the C-terminal actin-binding domain (C-ABD). E-Tmod29 can be generated by alternative splicing from an upstream promoter or by multiple transcriptional start sites from a downstream promoter. Promoter switching leads to a surge of E-Tmod41 in reticulocytes, which degrades quickly in the cytosol. We expressed recombinant isoforms in Escherichia coli and tested their binding toward TM5, G-actin, and F-actin. Solid-phase binding assays show that, without the N-terminal 102 residues, E-Tmod29 binds to TM5 or G-actin more strongly than E-Tmod41 does, but barely binds to F-actin after TM5 binding. Differential bindings explain the distinct localizations of E-Tmod29 in the cytosol and E-Tmod41 on the membrane. Sequential bindings and immunofluorescent staining further suggest that 1) TM5 binding to E-Tmod41 may open up the flexible N-terminal half, exposing N-ABD and unblocking C-ABD; 2) N-ABD binds to F-actin and C-ABD binds to G-actin; and 3) F-actin binding to N-ABD may prevent G-actin from binding to C-ABD. E-Tmod29 may thus modulate the availability of TM5 and G-actin for E-Tmod41 to construct the protofilament-based membrane skeletal network for circulating erythrocytes.
Yao
Weijuan
W
Department of Bioengineering, University of California, San Diego, California 92093-0412, USA.
Sung
Lanping Amy
LA
D000199:Actins / Q000235:genetics / Q000378:metabolism*; D017398:Alternative Splicing / Q000502:physiology*; D000818:Animals; D003599:Cytoskeleton / Q000235:genetics / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051379:Mice; D011401:Promoter Regions, Genetic / Q000502:physiology*; D011485:Protein Binding; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic / Q000502:physiology; D051348:Tropomodulin / Q000235:genetics / Q000378:metabolism*
D000199:Actins; D020033:Protein Isoforms; D011994:Recombinant Proteins; C495831:Tmod1 protein, mouse; D051348:Tropomodulin