Engineering Yeast for Functional Analysis of Human Cu Transport Proteins

Sammanfattning: ABSTRACT       Copper (Cu) is an important trace element that plays a vital role in several biological processes. Many proteins that are involved in various biochemical pathways require Cu as a cofactor. In human cells, Cu uptake is mediated by the high-affinity Cu uptake protein Ctr1, followed by the cytoplasmic Cu chaperone Atox1, which shuttles Cu from the plasma membrane to the Wilson disease protein ATP7B, a P1B-type ATPase located at the Golgi compartment. ATP7B incorporates Cu to Cu-dependent enzymes in the secretory pathway and also export excess Cu from the cells. Genetic defects in ATP7B may lead to a nonfunctional protein where Cu distribution is impaired, resulting in Wilson disease (WD). ATP7B is a multi-domain membrane-spanning protein with several protruding cytoplasmic domains. In contrast to its bacterial or yeast homologs, which have one or two metal-binding domains (MBD) respectively, ATP7B has six cytosolic MBDs. Many mechanistic questions around ATP7B function remain unanswered: e.g., the reason for as many as six MBDs and how Cu is released in the Golgi lumen.      In this thesis, the development of a new yeast model system for investigating Cu transport by human proteins is described. The system probes Cu shuttling from Atox1 to ATP7B, and within ATP7B, with yeast growth as the readout. Using this system, combined with biophysical methods such as NMR and MD simulations in some studies, we investigated the roles of the six MBDs in ATP7B (Paper I and III), examined Cu release by ATP7B in the Golgi (Paper II), and studied the effects on Cu transport due to MBD mutations causing WD (Paper IV). Our results provide new mechanistic information on how the six MBDs cooperate to move Cu along them. We also identified residues important for Cu release from ATP7B to the Golgi lumen. The collected results highlight the yeast model as a useful system for studying human Cu transport proteins, in particular when combined with other experimental methods. The yeast model system may be utilized to gain further understanding of various human Cu transport proteins as well as of disorders related to Cu mismetabolism, such as cancer and neurodegeneration.