To maintain polarity, epithelial cells continuously sort transmembrane proteins to the apical or basolateral membrane domains during biosynthetic delivery or after internalization. This thesis was designed to elucidate the molecular mechanism underlying protein trafficking. Our studies identified Rab13 as a critical GTPase regulator of biosynthetic cargos that are transported from the trans-Golgi network (TGN) to recycling endosomes before being delivered to the plasma membrane. We showed that Rab13 partially co-localizes with TGN38 at the TGN and transferrin receptors at recycling endosomes. Overexpression of dominant active or dominant negative alleles of Rab13 disrupted localization of the TGN marker, TGN38, in coverslip grown MDCK cells. Importantly, this phenotype was unique to Rab13, as compared to Rab8 and Rab10. In polarized MDCK cells, we microinjected cDNAs encoding Rab13 mutants and transmembrane cargo proteins. We found that mutations of Rab13 resulted in impaired surface delivery of cargos that normally sort to the plasma membrane through recycling endosomes (VSVG, A-VSVG, and LDLR-CT27). Rab13 mutants, however, had little effect upon cargos traveling a direct path to the plasma membrane (LDLR(Y18A), FcR, and HA). These results strongly support the hypothesis that Rab13 is involved in the transport of cargo from the TGN to the recycling endosomes.
A second part of this thesis examined the evolutionary conservation of sorting signals across different cell types. We characterized the sorting of a transmembrane protein, NgCAM that is expressed endogenously by both epithelial cells and neurons. We found that the same NgCAM cytoplasmic tail sequence (45-59) promotes enrichment in the axonal domain of neurons as well as apical localization in epithelial cells.
|Commitee:||Beitel, Greg, Horvath, Curt, Morimoto, Rick|
|Department:||Interdepartmental Biological Sciences Program|
|School Location:||United States -- Illinois|
|Source:||DAI-B 69/11, Dissertation Abstracts International|
|Keywords:||Membrane trafficking, Polarized epithelial cells, Rab13, Recycling endosomes|
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