Lucy Liu
Understanding Nuclear Transport: Localization of Importin Beta Proteins in Tetrahymena thermophila
Mentor:  Dr. Doug Chalker, Biology Department, Washington University in St. Louis

With the aid of importins and exportins, cytoplasmic proteins are able to efficiently travel across the nuclear membrane. Different types of cargo proteins contain unique nuclear localization signals that are recognized by specific importin alpha proteins (ImpA). These ImpA proteins contain an importin beta binding domain, to which an importin beta protein (ImpB) is able to attach. The ImpA-ImpB heterodimer is then able to guide movement of the cargo through the nuclear pore complex into the nucleus. Once inside the nucleus, Ran-GTP, a factor associated with ImpB, initiates the detachment of ImpA from ImpB and from the protein cargo. To continue this process of nuclear import, ImpA and ImpB are then recycled back into the cytoplasm.

Tetrahymena thermophila, a unicellular eukaryote, is an advantageous model organism for the dynamic study of nuclear transport because it contains two distinct nuclei, a macronucleus and a micronucleus. To begin to understand the specific targeting of nuclear proteins in T. thermophila, the Chalker Lab fused ImpA and ImpB genes to a green florescent protein (GFP) gene using a Gateway® recombination cloning strategy. The GFP allows one to visualize the locations of these ImpA proteins and ImpB proteins. The T. thermophila genome encodes for 11 putative ImpB genes. Throughout vegetation and conjugation, I examined the localization of ImpB #7, #9, and #10. ImpB #7 localized only to the micronucleus, ImpB #9 localized to both the macronucleus and to the micronucleus, and ImpB #10 localized to neither the micronucleus nor the macronucleus. Therefore, ImpB #7 and ImpB #9 appear to be importins. Since I saw no ImpB #10 localization to either of the two nuclei, ImpB #10 may not be an actual importin.

Although the Gateway® recombination cloning strategy provided an effective means of tagging ImpA and ImpB proteins, overexpressing these genes may result in artifactual localization of ImpA and ImpB proteins. Using homologous recombination, I attempted to incorporate the Micronuclear Linker Histone (MLH) gene tagged with a yellow florescent protein (YFP) gene into the T. thermophila genome to be transcribed under the control of its endogenous promoter. The MLH gene was inserted into a vector containing an YFP tag for visualization and a StuI restriction enzyme site to be used to direct homologous recombination. In addition, the construct contains a selectable marker that confers paromomycin once integrated into the genome. If homologous recombination occurred, the T. thermophila genome would be able to transcribe the MLH gene in its normal copy numbers. Moreover, this procedure could be used to tag ImpA and ImpB proteins in importin research. While I did not obtain any transformants, I hope to repeat this experiment, along with further localization studies of ImpB proteins in future research.