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Non-erythroid beta spectrin interacting proteins and their effects on spectrin tetramerization

Abstract

With yeast two-hybrid methods, we used a C-terminal fragment (residues 1697–2145) of non-erythroid beta spectrin (βII-C), including the region involved in the association with alpha spectrin to form tetramers, as the bait to screen a human brain cDNA library to identify proteins interacting with βII-C. We applied stringent selection steps to eliminate false positives and identified 17 proteins that interacted with βII-C (IPβII-C s). The proteins include a fragment (residues 38–284) of “THAP domain containing, apoptosis associated protein 3, isoform CRA g”, “glioma tumor suppressor candidate region gene 2” (residues 1-478), a fragment (residues 74–442) of septin 8 isoform c, a fragment (residues 704–953) of “coatomer protein complex, subunit beta 1, a fragment (residues 146–614) of zinc-finger protein 251, and a fragment (residues 284–435) of syntaxin binding protein 1. We used yeast three-hybrid system to determine the effects of these βII-C interacting proteins as well as of 7 proteins previously identified to interact with the tetramerization region of non-erythroid alpha spectrin (IPαII-N s) [1] on spectrin tetramer formation. The results showed that 3 IPβII-C s were able to bind βII-C even in the presence of αII-N, and 4 IPαII-N s were able to bind αII-N in the presence of βII-C. We also found that the syntaxin binding protein 1 fragment abolished αII-N and βII-C interaction, suggesting that this protein may inhibit or regulate non-erythroid spectrin tetramer formation.

Abbreviations

αII:

non-erythroid (brain) alpha spectrin

αII-N:

a recombinant protein consisting of the N-terminal region 359 residues of αII

AD:

activation domain of GAL4

βII:

non-erythroid (brain) beta spectrin

βII-C:

a recombinant protein consisting of residues 1697-2145 at the C-terminus of βII

BD:

binding domain of GAL4

IPαII-N :

proteins interacting with αII-N

IPβII-C :

proteins interacting with βII-C

pAD:

yeast twohybrid cloning vector pGADT7

pBD:

yeast two-hybrid cloning vector pGBKT7

pBR:

yeast three-hybrid cloning vector pBridge

QDO:

quadruple drop-out

SD:

synthetic defined

TDO:

triple drop-out

X-α-gal:

5-bromo-4-chloro-3-indolyl-α-galactopyranoside

YPDA:

yeast growth medium with yeast extract, peptone, dextrose and adenine

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Correspondence to Leslie W. -M. Fung.

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Sevinc, A., Fung, L.W.M. Non-erythroid beta spectrin interacting proteins and their effects on spectrin tetramerization. Cell Mol Biol Lett 16, 595 (2011). https://doi.org/10.2478/s11658-011-0025-9

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Key words

  • Brain beta spectrin
  • Spectrin tetramerization
  • Brain proteins
  • Yeast three-hybrid
  • Library screening
  • Spectrin interacting proteins