Abraham JK Koo and John B. Ohlrogge¹

Department of Plant Biology, Michigan State Universitty East Lansing, MI 48824-1325 USA
¹ To whom correspondence should be addressed. Phone (517)-3535-0611, fax: (517)-353-1926, e-mail: ohlrogge@msu.edu

Abstract
Conclusions
AtPEM library (Data of manuscript)

ABSTRACT

Plastid envelope proteins from the Arabidopsis thaliana nuclear genome were predicted using computational methods. Selection criteria were: first, to find proteins with NH2-terminal plastid targeting peptides from all annotated open reading frames from Arabidopsis thaliana, second, to search for proteins with membrane spanning domains among the predicted plastidial targeted proteins, and third, to subtract known thylakoid membrane proteins. 541 proteins were selected as potential candidates of the Arabidopsis thaliana plastid inner envelope membrane proteins (AtPEM candidates). Only 34% (183) of the AtPEM candidates could be assigned to putative functions based on sequence similarity to proteins of known function (compared to the 69% function assignment of the total predicted proteins in the genome). Of the 183 candidates with assigned functions, 40% were classified in the category of ‘transport facilitation’ indicating that this collection is highly enriched in membrane transporters. Information on the predicted proteins, tissue-expression data from expressed sequence tags (ESTs) and microarrays, and publicly available T-DNA insertion lines were collected. The data set complements proteomic based efforts in the increased detection of integral membrane proteins, low abundance proteins, or those not expressed in tissues selected for proteomic analysis. Digital northern analysis of ESTs suggested that the transcript levels of most AtPEM candidates were relatively constant among different tissues in contrast to stroma and the thylakoid proteins. However, both digital northern and microarray analysis identified a number of AtPEM candidates with tissue-specific expression patterns.

CONCLUSIONS

Plastids draw the attention of plant biologists in large part because of their defining roles in establishing the character of the plant cell. Envelope proteins may hold one key to the understanding of coordinated control between the plastid and the rest of the cell. So far there is no established inventory of plastid envelope proteins. In this study we attempted to predict integral plastid envelope proteins from the Arabidopsis thaliana nuclear genome using computational methods.  As the word ‘candidates’ implies the results of our study are not definitive due to the nature of prediction software and due to the possibility of errors in protein annotations in the genome databases (van Wijk, 2001). Although it is likely that at least 10% of the AtPEM candidates represent incorrect predictions, it is reasonable to assume that the selected candidates represent a large portion of the real envelope proteome. These candidates can be used as a starting point for designing further biological experiments. For example one alternative approach to complement ‘proteomics’ approaches is to experimentally characterize envelope localization of selected candidates. The AtPEM candidates could be analyzed for their targeting to the chloroplasts and partitioning to the envelopes by in vitro reconstitution of import into the chloroplast followed by fractionation into the suborganellor compartments. This method can be especially powerful in identifying highly-hydrophobic, low-expressed transporters and also can circumvent the difficulties in isolation of pure plastid envelope from many nonphotosynthetic tissues. This approach is currently being developed and evaluated in our laboratory. Another application to further characterize selected candidates will be to define subsets of AtPEM proteins that show concerted spatial, developmental, or conditional expression profiles. Finally, based on our database, and such selections, a more focused analysis of the phenotypes and biochemical compositions altered in T-DNA insertion lines can be carried out.

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