LOCALIZATION OF PROTEIN CHIMERAS THAT INCLUDE GREEN FLUORESCENT PROTEIN (GFP)

绿色荧光蛋白（GFP）已被证明是巨大的

The green fluorescent protein (GFP) has proven to be tremendously valuable in providing cell biologists with a marker that gives a robust signal in vivo but is largely non-destructive to the proteins with which it is fused. In yeasts and other genetic organisms it is often possible to replace an endogenous gene with a version, fused with the GFP, and show that wild type function is fulfilled by this "tagged" allele. When expressed under the gene's normal promoter, artifacts of over-expression are also avoided. We have found that an affinity purified, polyclonal antibody raised in rabbits against the GFP by Kahana will recognize its antigen with a S/N of ~200, even after the rigors of our fast freezing/freeze-substitution fixation (see Subproject T12). Employing these preparation methods, Morphew has now localized the kinesin-like proteins, Kar3p and Kip2p in budding yeast. She has also localized a novel protein whose mutation is synthetically lethal with kar3-delete, Sms1p. The results are proving extremely interesting to students of the yeast cytoskeleton and mitosis, because our results have already cast doubt on several hypotheses about motor position in vivo. We are also sharing this technology with Mark Winey's, Trisha Davis', and Karl Ekwall's labs, and they are using it for several additional yeast protein localizations. C11