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United States Department of Agriculture

Agricultural Research Service

Title: DECODING PROTEIN LOCALIZATION

Authors
item Stockman, Kevin - SOUTHWEST MO STATE UNIV
item Mooney, Brian - UNIV OF MISSOURI-COLUMBIA
item Miernyk, Jan

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: June 19, 2000
Publication Date: N/A

Technical Abstract: A goal of "functional genomics" is to assign a biological role to all of the proteins identified during whole-genome sequencing projects. To better understand the function of these proteins, it is essential to know their subcellular location. The J-domain proteins of the model plant Arabidopsis thaliana comprise a surprisingly large and complex family; to date more than 30 members have been identified. The amino acid sequences of these proteins have been analyzed using the subcellular localization algorithms: PSORT and TargetP. In several instances, multiple subcellular locations were predicted. Analysis of the potential localization signals used plasmid-driven, coupled in vitro transcription/translation. Translation incorporated 35S-methionine into the proteins. Products were analyzed by SDS-PAGE plus autoradiography. In some experiments, the products were post-translationally incubated with isolated, intact pea (Pisum sativum) seedling chloroplasts. Import into these organelles was determined by conversion to a smaller form that is resistant to protease digestion. Nuclear import could not be directly tested in the limited time period, so an indirect assay was devised. A. thaliana importin-alpha was engineered to include six successive histidine residues at the N-terminus. The (His6) importin-alpha was isolated from transformed bacteria, and specifically bound to Ni-NTA agarose. The immobilized recombinant protein was then used as an affinity-column to test binding of the 35S-labeled translation products.

Last Modified: 8/31/2014
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