Intracellular Processing and Assembly of Fet3p

All multicopper oxidases are glycosylated molecules that are either secreted or membrane bound. These molecules enter the secretory pathway as nascent polypeptides and are subsequently processed in the endoplasmic reticulum and Golgi apparatus. The assembly of an active Fet3p requires the addition of copper to the apo form to generate the active holo enzyme. Our understanding of the genes required for the copper loading of apoFet3p have been furthered by intense work that has been done defining the molecular basis of Menkes and Wilson's disease (2,29). The genes responsible for these disorders are highly homologous ATP-copper transporters that are localized in post-Golgi vesicles. Like many of the genes involved in copper metabolism, there is a yeast homolog, termed CCC2. Genetic studies demonstrated that Ccc2p is the protein required for the copper loading of Fet3p (30). In the absence of Ccc2p, an apoFet3 is transported to the cell surface. The apoFet3p lacks ferroxidase activity and is unable to mediate high-affinity iron transport. Ccc2p is found on a post-Golgi vesicle and it is thought that it is in this vesicle that apoFet3p obtains its copper. Exactly where or what this post-Golgi vesicle is has been clarified by studying the vacuolar assembly, an organelle that is analogous to the mammalian lysosome.

Fig. 4. Model for the intracellular processing of holoFet3p. CPY is a soluble vacuolar protein and ALP is a vacuolar membrane protein. These two proteins as well as apoFet3p and Ftrlp are synthesized in the rough endoplasmic reticulum and then transferred to the Golgi, where glycosylation takes place. apoFet3p is transferred from the Golgi to a vesicle that contains Ccc2p and Geflp. It is in this vesicle that the copper is added to apoFet3p forming the holo enzyme. It is thought that CPY and ALP might also pass through this compartment. Mutations in genes that disrupt vesicular traffic at a post-Golgi step results in the transfer of apoFet3p, ALP, and CPY to the cell surface. The soluble enzyme CPY is secreted into the media, whereas apoFet3p stays on the cell surface. ALP contains endocytic signals that permit it to be internalized to the vacuole.

Fig. 4. Model for the intracellular processing of holoFet3p. CPY is a soluble vacuolar protein and ALP is a vacuolar membrane protein. These two proteins as well as apoFet3p and Ftrlp are synthesized in the rough endoplasmic reticulum and then transferred to the Golgi, where glycosylation takes place. apoFet3p is transferred from the Golgi to a vesicle that contains Ccc2p and Geflp. It is in this vesicle that the copper is added to apoFet3p forming the holo enzyme. It is thought that CPY and ALP might also pass through this compartment. Mutations in genes that disrupt vesicular traffic at a post-Golgi step results in the transfer of apoFet3p, ALP, and CPY to the cell surface. The soluble enzyme CPY is secreted into the media, whereas apoFet3p stays on the cell surface. ALP contains endocytic signals that permit it to be internalized to the vacuole.

Genetic screens have led to the identification of many genes required for the transport of vacuolar components, both vacuolar enzymes and membrane components. Mutants defective in vacuole formation are termed vps or vma mutations (31). Many of these mutants show an inability to grow on low-iron media (32,33). This defect results from a defective high-affinity iron-transport system. The Fet3p delivered to the cell surface is not copper loaded and lacks ferroxidase activity. Golgi-derived vesicles containing appropriately glycosylated proteins, including apoFet3p and the vacuolar enzyme carboxypeptidase Y (CPY), are delivered to the cell surface instead of the post-Golgi vesicle that normally houses the machinery for copper loading or the vacuole that normally houses CPY. These results position the Ccc2p vesicle between the trans-Golgi and the vacuole (Fig. 4).

In addition to the VPS genes, other genes are required for the copper loading of apoFet3p. One group of genes encodes subunits of the vacuolar H+-ATPase (34). In the absence of a subunit of the enzyme, vacuoles and presumably other post-Golgi prevacuolar compartments are not acidified. A second gene encodes a member of the family of voltage-regulated chloride channels (35). The yeast gene GEF1 is highly homologous to the human gene CLC5, which encodes a vesicular chloride transporter that is specific to the kidney (36). The yeast gene also encodes a vesicular protein that has been localized to the same vesicle as Ccc2p (37,38). Defects in gef1 also result in the presence of apoFet3p on the cell surface (38,39).

A hypothesis has been presented to explain why a defect in a chloride channel would result in an apoFet3p (38). In a membrane-bound vesicle, the introduction of cations such as Cu+ and H+ would quite rapidly result in an unfavorable membrane potential that prevents further introduction of cations, thereby limiting copper concentration and pH. Under normal conditions, this potential difference effects the opening of the voltage-gated chloride channel. Chloride enters the vesicle as a result of the electrical-chemical gradient. The introduction of anions into the vesicle neutralizes the positive potential difference resulting from cation accumulation. When the voltage potential has been dissipated, the chloride channel would close. The operation of the channel prevents the formation of an unfavorable gradient, permitting a high copper concentration and reduced pH. Disruption of chloride channels results in dysregulated vesicular ion homeostasis, reducing vesicular copper concentration and increasing vesicular pH. This hypothesis implies that there is a pH dependency to the copper loading of apoFet3p. To test this prediction, Davis-Kaplan et al. developed an in vitro system to measure the pH dependency of copper loading (39). The system employed a mutant strain lacking Ccc2p. As mentioned earlier, in the absence of Ccc2p, an apoFet3p is placed on the cell surface. Cell-surface apoFet3p can be converted to holoFet3p at 0°C, suggesting that no metabolic energy is required. The assay for copper loading of apoFet3p is to expose cells to copper at different pH's at 0°C, wash the cells and then incubate them in copper-free buffers at 30°C to assay iron transport. The results of this experiment demonstrated that the copper loading of apoFet3p was pH dependent. The greatest degree of copper loading occurred at pH 4.5. This result confirms the role of the H+-ATPase in the maturation of Fet3p. The experiment, however, revealed an unusual feature in that copper loading of apoFet3p was chloride dependent. Without chloride, little copper loading occurred at all pH's examined. Chloride could be replaced by bromine and to a lesser extent by iodine, but not by other nonhalide anions. The requirement for chloride to load apoFet3p was confirmed by a second assay. holoFet3p as a multicopper oxidase can be assayed using organic substrates. An apoFet3p extracted from cells with a CCC2 deletion has, as expected, no enzymatic activity on organic substrates. An apoFet3p copper loaded in vitro shows enzymatic activity. Using this assay, the copper loading of Fet3p was again shown to be chloride dependent.

The specific role of chloride in the loading of Fet3p is unknown. Trivial explanations such as chloride is required to maintain copper in solution have been ruled out. Examination of the kinetics of the chloride dependence of copper loading has shown an allosteric interaction between copper and chloride. In the presence of different concentrations of chloride, there is a sigmoidal relationship between copper and chloride. When chloride concentration is high, lower concentrations of copper effect the formation of a holoFet3p. The converse is also true, when copper concentration is high, lower concentrations of chloride are required to copper load Fet3p.

Examination of the literature reveals a precedent for chloride requirement for the metallation of a protein. The yeast enzyme leucine aminopeptidase 1, the product of the LAP1 gene, is a zinc-containing vacuolar exopeptidase. This enzyme requires both zinc and chloride for activity. In the absence of chloride, zinc will not bind to the enzyme. It was found that chloride bound to the enzyme, but only in the presence of zinc (39,40). These studies showed a direct requirement of chloride for the metallation of the enzyme. At present, it has not been demonstrated that chloride binds to Fet3p. Further, the studies on chloride binding do not rule out the hypothesis that disruption in the chloride channel prevents the formation of a holoFet3p by affecting ion homeostasis. It may well be that both disruption of iron homeostasis and the lack of chloride required for the direct metallation of the protein are responsible for the inability of GEF1 -deletion strain to copper load apoFet3p.

The study of FET3, its biochemistry and physiology, has provided insight into both the mechanism of iron transport and the physiology of copper. As copper homeostasis is required for Fet3p activity, analysis of Fet3p function has led to the discovery of genes involved in copper metabolism. Further, analysis of the assembly of Fet3p has also led to insight into human iron metabolism. Finally, as the oxidase/permease iron-transport system may be a virulence factor for pathogenic fungi, the transport system them becomes a target for therapeutic approaches.

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