Due in large part to the clarity that yeast genetics brings to the analysis of protein function, is an excellent model organism for studying many fundamental processes of eukaryotic cells (15).The importance of oxidative metabolism to eukaryotic cells has led to conservation of mechanisms of copper metabolism from yeast to human cells.Ccc2 is required for transport of copper from the cytosol into the lumen of the trans-Golgi network, where the multi-copper ferroxidase Fet3, the yeast homolog of ceruloplasmin, is loaded with copper (20, 21).The yeast genome does not encode a PHM or a PAL homolog.
P-type ATPases are likely to play a role in copper transport into the secretory pathway.The resulting lack of copper compromises the function of many tissues, resulting in death in early childhood.Mutations in the ATP7B gene cause Wilson disease, which is characterized by failure of the liver to excrete copper into the biliary tract or to deliver copper to ceruloplasmin, a multi-copper oxidase needed for serum iron homeostasis (13).In the present study, we determined whether active PHM could be produced in the yeast secretory pathway and whether its production requires the assistance of known copper transporters and chaperones.In particular, the roles of the P-type ATPase Ccc2 and the cytosolic copper chaperone Atx1 were investigated.Similarly, production of active PHM in mouse fibroblasts is impaired in the presence of a mutant oxidase is essential for respiration, superoxide dismutase is essential for free radical detoxification, lysyl oxidase is essential for maturation for connective tissue, ceruloplasmin is essential for iron uptake, tyrosinase is essential for melanin synthesis, and dopamine β-monooxygenase is essential for catecholamine formation (1).Another important copper-dependent enzyme, peptidylglycine α-amidating monooxygenase (PAM), catalyzes the C-terminal amidation of glycine-extended peptide precursors, a modification essential for the bioactivity of numerous hormones and neuropeptides (2-5) (see Fig. Peptide amidation occurs in two consecutive steps that require the peptidylglycine α-hydroxylating monooxygenase (PHM) and peptidyl-α-hydroxyglycine α-amidating lyase (PAL) domains of the bifunctional PAM protein (see Fig. This reaction can be initiated in the trans-Golgi network but occurs primarily within secretory granules (6, 7). The PHMcc protein expressed in mammalian CHO cells and the α-mating factor-PHMcc fusion protein expressed in wild-type yeast cells (YPH252) are shown.We examined the roles of yeast Ccc2, a P-type ATPase related to human ATP7A (Menkes disease protein) and ATP7B (Wilson disease protein), as well as yeast Atx1, a cytosolic copper chaperone.We expressed soluble PHMcc (ore) in yeast using the yeast pre-pro-α-mating factor leader region to target the enzyme to the secretory pathway.The yeast α-mating factor precursor is diagrammed with α-factor peptides ( Production of amidated peptides by PAM requires copper and molecular oxygen and two reducing equivalents, usually supplied by two molecules of ascorbate.The PHM domain of PAM binds copper at two non-equivalent sites (Cu), both of which are critical for activity (8).