AFFINITY PURIFICATION OF ORF256-COXI RNA BINDING PROTEINS FROM WHEAT (Triticum aestivum) MITOCHONDRIA

Document Type : Original Article

Authors

1 Current address: Department Genetics, Faculty of Agriculture, Alexandria University, Alexandria, Egypt

2 Department of Biochemistry, Kansas State University, Manhattan, Kansas 66506, USA

Abstract

Cytoplasmic male sterility (eMS) is associated with the presence and
expression of the orf256 gene in the hybrid plants between Triticum aestivum (Ta) and
T. timopheevi (Tt). The gene is upstream of coxl gene in T. timop < /em>heevi mitochondrial
DNA (mtDNA). whereas it is not present in T. aestivum mtDNA. It is expressed with
cox I in the same mRNA in T. timop < /em>heevi, fertility restored lines (FR). and eMS lines.
Expression as a 7 kD protein occurs only in CMS lines. Fulllength-(3.1 kb) transcripts
as well as processed transcripts with 5' termini within the orf256 coding region have
been detected in both eMS and FR lines. In T. timopheevi mitochondria. there is only
a trace of full-length 3.1 kb transcript, and major transcripts have 5' termini within the
orf256 coding region. This prevents any significant expression of the orf256 protein
product. Orf256-coxl RNA binds many proteins in mitochondrial extracts from CMS.
Ta, FR, and Tt, but binds to 42 and 39 kD proteins from Ta extract only (EI-Shehawi
et. aI., 2003).
We used orf256-coxl mRNA to test the effects of phosphorylation as well as
phosphatase inhibitors (sodium vanidate) on the RNA binding to mitochondrial
proteins. In addition, the RNA was used in affinity purification experiment to isolate
RNA binding proteins from Ta mitochondrial extract. In this report, we conclude that
binding of orf256-coxl RNA to mitochondrial proteins is not dependent on the 5'- or 3'-
untranslated regions of the RNA. The phosphorylation state of mitochondrial proteins
appears to affect their binding to the RNA. The 42 and 39 kD proteins fractionate in
the 0-20% ammonium sulphate fraction. Six proteins were affinity purified from the 0-
20% fraction with biotin-labeled RNA. N-terminus sequencing revealed that the 42 kD
protein is similar to the coatmer complex epsilon chain protein of Arabidopsis thaliana. 

Keywords

Journal of Plant Production
Volume 29, Issue 3
March 2004
Pages 1215-1226

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