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A structure/function analysis of zinc efflux proteins in Drosophila melanogaster

thesis
posted on 28.02.2017, 01:21 by Dechen, Kesang
The importance of zinc homeostasis in the human body is becoming increasingly apparent with all the current findings on zinc-associated disorders. There are a number of diseases and syndromes associated with impaired zinc status such as Acrodermatitis Enteropathica, Alzheimer's disease, and diabetes to name a few. The study of how zinc homeostasis is maintained is a current research topic that is gaining more significance: Mammals have zinc influx and efflux proteins, namely ZiPs and ZnTs, that work in a coordinated fashion to maintain zinc homeostasis by bringing zinc into or taking it out of the cytoplasm respectively. In humans, there are at least 14 ZiP and 10 ZnT transporters that may function to maintain zinc homeostasis. In Drosophila, there are 10 putative Zips and 7 ZnTs that were identified by amino acid sequence homology to the human zinc transporters. The Drosophila model system, being a much easier model system to manipulate genes and study their function, will be utilized in the current study for a structure / function analysis of key putative Drosophila zinc efflux transporters and the in-depth functional characterization of one particular fly ZnT gene. The first part of this thesis consists of a search for the presence of localization signals in the Amino or Carboxyl domains of four Drosophila ZnTs. The sub-cellular localization of hybrid and truncated dZnTs was examined by overexpressing mCherry-tagged fusion proteins in the larval salivary gland cells. Inferences on the presence of localization signals were made by observing if certain Amino or Carboxyl termini directed the protein to specific sites. The functionality of these hybrid and truncated ZnT proteins was tested using a zinc toxicity eye phenotype. Our results suggested the presence of a localization signal in the carboxyl termini, which can be activated by signals from the amino termini of the protein. The nonfunctionality of the hybrid proteins suggested that the proteins may function by the formation of dimers. The next part of the thesis consists of an investigation into the function of dZnT86D, a zinc efflux protein that is homologous to mammalian ZnTs 5 and 7. Overexpression and suppression studies in various tissues revealed a role for this protein in the eye, ring gland,thorax and neural tissues. Localization studies indicated that dZnT86D may be localized to the Golgi or the endoplasmic reticulum. The zinc-modulated rescue of a dZnT86D overexpression eye phenotype suggested that this protein functions as a zinc efflux protein. Genetic interactions with other zinc transporters suggested that the zinc transporters work in conjunction to maintain cellular zinc homeostasis. A dZnT86D null mutant was generated and null mutant analysis suggested that this gene is essential for viability after the second larval instar stage and may have a role in the activity of alkaline phosphatase. The regulation of this gene is tightly controlled as suggested by the failure of our overexpression constructs to rescue the recessive larval lethality. Further analysis into this gene is required to determine its endogenous expression patterns and sub-cellular localization. The exact role dZnT86D might play during development would be an interesting avenue to follow. This null mutant background could be a used as a tool for further analysis of the other zinc transporters in Drosophila as well as mammalian zinc transporters.

History

Campus location

Australia

Principal supervisor

Richard Burke

Year of Award

2013

Department, School or Centre

Biological Sciences

Degree Type

RESEARCH_MASTERS

Faculty

Faculty of Science