Date of Award

3-27-2019

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Biology

First Advisor

John Bush

Abstract

Localization and functional analysis of the small monomeric GTPase RabY in the unicellular amoeba Dictyostelium discoideum was investigated. I constructed 2 cell lines that overexpressed GFP tagged RabY (OE) and expressed a dominant negative form of GFP-RabY (T25N). Sequence analysis revealed homology to Rab1a genes in several organisms (ER/Golgi localized in humans). Therefore, I hypothesize the GFP tagged RabY protein is within the Golgi and/or Endoplasmic Reticulum. Analysis using immunofluorescence, quantitative and other techniques revealed GFP-RabY (OE) is localized at the Dictyostelium Golgi apparatus and within the endosomes. However, the GFP-RabY (T25N) protein was found to be dispersed intracellularly. Thus, RabY localizes and probably functions in trans-Golgi (TGN). Microscopic analysis revealed that GFP-RabY (T25N) had an aberrant endocytic distribution using TRITC dextran marker. The secretion of two lysosomal enzymes, acid phosphatase and α- mannosidase was lower in both RabY cell lines. The overexpressed GFP-RabY protein also partially co-localized with the contractile vacuoles (CV) and so did the dominant negative GFP-RabY (T25N) protein. However, compared to other CV localized Dictyostelium Rab protein functions, RabY (T25N) expression did not alter function of the CV under osmotic stress. Finally, RabY function was studied during the Dictyostelium 24-hour developmental cycle where 100,000 cells aggregate and form a stalk with spores. The Golgi during Dictyostelium feeding cycle is not in the traditional stacks. Golgi associated glycosylation, glucose transferases and proteolytic processes occurring in the Golgi have impacts on development and the nature of the Golgi itself in several organisms (Mironov 2010, Zhong 2011). Therefore, because of RabY’s association with Golgi, it may function in development as well as feeding life stages. One major result found was that RabY (OE) cell line had small developmental fruiting body structures as well as having an increased velocity toward the Dd chemoattractant, cAMP. Thus, RabY functions in chemotaxis leading to premature aggregation and abnormal fruiting bodies. While more RabY functional investigations remain, my work is the first description of RabY in Dictyostelium highlight areas for further research.

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