Development of a Yeast Model for Functional Analysis of Human Copper Transport Proteins

Licentiate thesis, 2018

ABSTRACT

Copper (Cu) is an important trace element that plays a vital role in several biological processes. It mediates numerous biochemical functions to maintain cellular homeostasis making it an essential metal for sustaining life. Many proteins or enzymes that are involved in various biochemical pathways require copper as a cofactor (also a regulator).

In human cells, the Cu uptake is mediated by high-affinity copper uptake protein (Ctr1), followed by cytoplasmic chaperone Atox1 that shuttles Cu from the plasma membrane to Wilson’s disease protein ATP7B (P-type ATPase), a membrane-bound protein located at the Golgi apparatus. ATP7B incorporates Cu to various Cu-dependent enzymes in the secretory pathway. The main biological role of ATP7B (or Wilson Disease Protein) is to maintain the copper balance inside the human cell. Genetic defects in ATP7B often leads to a nonfunctional protein where copper balance is impaired and this condition results in Wilson’s disease (WD).  ATP7B is a large multi-domain membrane transport protein that shows typical characteristics of a P1b type ATPase. In contrast to its bacterial (CopA) or yeast (Ccc2) counterparts which have one or two metal binding domains (MBD) respectively, the human ATP7B has six cytosolic MBDs in the N-terminal region. The reason for the presence of these six MBDs in ATP7B is not completely understood, and neither is the ATP7B mediated copper release in the Golgi.

In this thesis, the development of a novel yeast model system for investigating the functional role of ATP7B in copper transport is described. The system probes shuttling of copper via human Atox1 to ATP7B proteins when expressed in a yeast humanized model. Using this system, we investigated the roles of six metal binding domains (MBDs) in ATP7B (Paper 1) and examined the Cu release (paper II). The results address the importance of the yeast model for studying human Cu transport proteins, the role of MBDs in ATP7B mediated Cu transport, the role of Atox1 in shuttling Cu and the significance of the luminal loop in ATP7B for Cu release function. Overall, the yeast model system developed in this thesis has great future potential for studying human copper transport proteins, which are involved in genetic diseases such as WD. The designed system can be expanded by using system biology approaches, to gain further understanding on human copper transport as well as copper transport related disorders.