© 1995 Oxford University Press
RESEARCH-ARTICLE |
Purification, sequencing and functions of calreticulin from maize
1Horticulture Research International Wellesbourne, Warwick CV35 9EF, UK
2Department of Biological and Molecular Sciences, Oxford Brookes University Oxford 0X3 OBP, UK
3Department of Plant Sciences, South Parks Road, Oxford University Oxford 0X1 3RB, UK
4To whom correspondence should be addressed. Fax: +44 1789 470552.
The most abundant proteins in the lumen of the endoplasmic reticulum (ER) are thought to be molecular chaperones, some of which might also be involved in calcium storage and release. We have purified calreticulin from maize by ion exchange and reverse-phase chromatography. Identity with plant and animal calreticulins was confirmed by N-terminal amino acid sequencing and it was shown to bind calcium with a calcium overlay technique. An antiserum raised to the purified protein was used to screen an expression library and the full coding sequence for maize calreticulin was determined from the clones selected. The sequence shows 96% identity to barley calreticulin and 55% identity to animal calreticulins. The three major functional regions are conserved, as are targeting and retention features. When visualized by indirect immunofluorescence microscopy, calreticulin was found to be confined to the ER and nuclear envelope of maize root cells. It was distributed throughout the ER compartment and we found no evidence of calreticulin- enriched areas of ER, such as might be associated with specialized calcium storage domains. Increasing or decreasing extracellular calcium did not induce measurable changes in calreticulin levels. In addition, maize calreticulin, as well as other recognized chaperones, was shown to bind to denatured protein and could be eluted specifically by nucleoside trisphosphates.
Key words: Endoplasmic reticulum, calcium-binding protein, immunofluorescence, targeting, Zea mays L
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