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First published online June 26, 2009; 10.1105/tpc.109.068668

The Plant Cell 21:1632-1636 (2009)
© 2009 American Society of Plant Biologists

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PERSPECTIVE

Validating the Location of Fluorescent Protein Fusions in the Endomembrane System

Ian Moorea and Angus Murphyb,1

a Department of Plant Sciences, University of Oxford, Oxford OX1 3RB, United Kingdom
b Department of Horticulture, Purdue University, West Lafayette, Indiana 47907

1 Address correspondence to murphy{at}purdue.edu.

ABSTRACT

Assessment of gene function generally requires knowledge of the sites of action of gene products. Several experimental approaches can provide relevant information, but all have their limitations and the potential for experimental artifact. In this article we focus on the endomembrane organelles and on the methods that can be used to validate the location of fluorescent protein fusions. We discuss the utility of redundant localization techniques, complementation of mutant phenotypes, and integration of localization data with expected biological function as methods to achieve consensus. We argue that no single piece of evidence is sufficient to address the issue, and that all approaches can reveal useful information about the true steady state location of a protein or about other aspects of its transport and dynamics. As ever, the critical point is the subjective interpretation one puts on each observation in light of the experimental conditions and other pertinent data. We illustrate these points with some successes and failures in our own work.




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[Abstract] [Full Text] [PDF]


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