Erythrocyte Tropomodulin Isoforms with and without the N-terminal Actin-binding Domain

Abstract

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 ϳ37 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. Erythrocytes are responsible for the transport of O 2 and CO 2 in tissues throughout the body. Their cell membranes have a thin layer of protein skeleton under the lipid bilayer. The viscoelasticity and durability of this protein skeleton allows erythrocytes to circulate for 120 days in humans and 60 days in mice. These membrane skeletal proteins are differentially expressed during erythroid differentiation, and the complex transcellular cytoskeleton is remodeled into a membrane skeleton during reticulocyte maturation (1, 2).The membrane skeletal network consists of a large number of basic repeating units interconnected to each other. Each unit contains two kinds of major complexes, a junctional complex (JC) 3 and suspension complex (SC), located in the center and up to six in the periphery, respectively (Fig. 1). A JC includes actin, tropomyosin (TM), erythrocyte tropomodulin (E-Tmod), protein 4.1R, adducin (3), protein 4.9 (dematin), and p55 (for review see Refs. 3 and 4), with six spectrin heterodimers (Sp) radiating from the center. Associated near the distal end of each Sp is an SC. An SC consists of several proteins, including anion exchanger (band 3), ankryin, and protein 4.2, which suspends the skeletal network to the lipid bilayer (for review see Refs. 5 and 6).Some of these membrane skeletal proteins are encoded by genes known to have alternative splicing, alternative promoters, multiple transcriptional start sites, and/or multiple polyadenylation sites (7-14). Indeed, some in JC are cell type-specific isoforms generated by one or more of the above mechanisms. For example, exon 16 of protein 4.1R, which encodes the 8-kDa spectrin/actin-binding domain (ABD), is alternatively spliced (15, 16). ␤ spectrin, which has an ABD, is generated by the differential splicing of its exon 32 (9). Interestingly, ␣ spectrin, which has no ABD, is synthesized three times more than ␤ spectrin, but is quickly degraded in reticulocytes if not incorporated into the membrane skeleton (1...

Mechanobiology of Erythrocytes from Adult Mice Homozygous for a Targeted Disruption of the E-Tmod Gene at Exon 1

“…30 Figure 3c revealed that all three genotypes were indeed able to form dimers. Image J was used to quantify the ratio between the dimer and monomer (58/29).…”

“…This finding suggests that there may be another promoter upstream from exon 3, generating a transcript which uses the AUG in exon 3 to translate E-Tmod29. Previously reported mechanisms of alternative splicing using transcripts starting from exon 0 or multiple transcription starts sites at exon 1 30 are not able to generate transcripts encoding E-Tmod29 in E-Tmod À/À cells, because such transcription would have been terminated at the end of the lacZ gene. Since this homozygous E-Tmod E1 KO mouse model still expresses the short isoform E-Tmod29, it is now clear that our E-Tmod À/À mouse model has an E-Tmod41 null mutation, but does not have an E-Tmod29 null mutation.…”

“…We investigated both the ghost membrane and whole cell lysates because E-Tmod41 is incorporated into the membrane skeleton and E-Tmod29 remains in the cytosol. 30 Therefore, the Western blot analysis of ghost membranes could only reveal the presence or absence of E-Tmod41 in different genotypes, while the analysis of whole cell lysates could reveal potentially both cytosolic E-Tmod29 and membrane-bound E-Tmod41. Therefore, the analyses of the ghost membrane and the whole cell lysates are needed to detect different expression patterns of these two isoforms in different compartments among three genotypes.…”

“…30 It is not clear whether E-Tmod29, which has its AUG initiation codon in exon 3, is expressed in Tmod À/À erythrocytes. Therefore, SDS-PAGE and Western blot analysis were also performed on whole erythrocyte lysates which contain both membrane and cytosol.…”

“…13 We discovered a short isoform of 29 kDa (E-Tmod29) derived from the mouse E-Tmod gene. 30 E-Tmod29 lacks the N-terminal F-actin-binding domain but retains the C-terminal G-actin-binding domain. E-Tmod29 can be generated by alternative splicing from an upstream promoter (P E0 , 5¢ to exon 0) or by multiple transcriptional start sites from a downstream promoter (P E1 , 5¢ to exon 1).…”