Validating the location of fluorescent protein fusions in the endomembrane system.
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.
Moore
Ian
I
Department of Plant Sciences, University of Oxford, Oxford OX1 3RB, United Kingdom.
Murphy
Angus
A
D004591:Electrophoresis, Polyacrylamide Gel; D005816:Genetic Complementation Test; D007150:Immunohistochemistry; D007425:Intracellular Membranes / Q000378:metabolism* / Q000648:ultrastructure; D008164:Luminescent Proteins / Q000032:analysis*; D009154:Mutation; D015388:Organelles / Q000378:metabolism; D010641:Phenotype; D010940:Plant Proteins / Q000032:analysis* / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000032:analysis*; D012107:Research Design
D008164:Luminescent Proteins; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins