Bourbouloux et al. [5
] previously demonstrated that Hgt1p is quantitatively the major GSH uptake transporter in S. cerevisiae.
Without this transporter, the cells have minimal ability to take up GSH from the culture medium and show marked growth retardation when GSH is the only sulfur source. However, hgt1-Δ cells do retain some ability to take up GSH at a slow rate (e.g., see Fig. ), and to grow slowly using GSH as a sulfur source (Fig. ). Thus, yeast cells have an alternate mechanism for taking up GSH, although this alternative mechanism has not been identified at the molecular level and has not been characterized biochemically.
The present study identifies five human genes, PMEPA1, LAPTM4α, SLC25A1, LITAF, and CYYR1 that can functionally complement the growth defect of hgt1-Δ cells cultured in a medium in which GSH is the only sulfur source. Each of these genes encodes a protein containing PY motifs that are thought to be important for regulating protein cell surface expression via interacting with the ubiquitin ligases NEDD4/Rsp5p [14
]. The first PY motif was identified through a functional cDNA library screen for putative ligands of the WW domain of the Yes-associated protein [18
]. The presence of these motifs allows the PY motif-containing proteins to bind to WW domains in target proteins, including the E3 ubiquitin ligase NEDD4 [19
]. NEDD4 protein belongs to the Hect-domain family of E3 ubiquitin-protein ligase. A major function of such proteins is to down-regulate the cell surface expression of membrane proteins via ubiquitin-dependent endocytosis, followed by degradation in both proteasomes and lysosomes [14
]. Mutations in the PY motifs of the epithelial Na+
channel disrupt its interaction with NEDD4, which leads to constitutively increased channel activity and hypertension in the Liddle syndrome [21
]. Rsp5p is the unique yeast homologue of NEDD4. The direct interaction between Rsp5p and its target protein through PY-WW binding is essential for multiple-vesicular body targeting and endosomal sorting of several yeast membrane proteins [22
] Indeed, some studies have also indicated possible associations of NEDD4 with PMEPA1 [24
], LAPTM4α [25
], and LITAF [26
]. Regulation of plasma membrane protein turnover by this mechanism may either occur directly, by binding of Rsp5p/NEDD4 to specific membrane proteins that contain PY motifs, or indirectly, by binding of Rsp5p/NEDD4 to PY-containing adapter molecules that then target the ubiquitin ligase activity to specific plasma membrane proteins [17
The present results are consistent with a model in which these five human genes enhance GSH uptake by increasing the activity of an undefined yeast low affinity GSH uptake mechanism [5
]. As illustrated in figure 5, the enhanced [3
H]GSH uptake in cells expressing the five human genes was uniformly inhibited by high concentrations of unlabeled GSH, GSSG, and ophthalmic acid, suggesting that the effects of these genes on [3
H]GSH uptake involves a common low affinity uptake pathway. Although the mechanism by which these genes enhance the activity of this alternate mode of GSH uptake is unknown, they may do so by increasing production, intracellular targeting, or the stability of components of the uptake machinery. One possibility is that expression of the PY-containing genes titrates the Rsp5p-associated degradation pathway away from some endogenous yeast transporter, thereby enhancing its activity. The enhanced turnover of general endocytotic markers detected in cells expressing the PY-containing genes might then reflect increased turnover of membrane proteins via an alternative latrunculin-A-insensitive pathway.
By analogy, the PY-containing protein Ndfip2 appears to increase cell surface expression of the amiloride-sensitive epithelial sodium channel (ENaC) by interfering with the Nedd4-mediated regulation of ENaC [28
]. Ndfip2 contains three transmembrane domains and two amino terminal PPxY motifs, which physically interact with the WW domains of Nedd4 family proteins. However, this stimulatory effect of Ndfip2 on ENaC activity appears to be relatively selective, as Ndfip1, a highly related protein that also contains PY motifs, has no effect on ENaC activity [28
]. In addition, Ndfip2 did not increase the transport activity of CFTR or Kir1. 1a, indicating selectivity for the target proteins. In the present study, expression of Ndfip2 in yeast cells did not produce stimulatory effects in GSH uptake, but this negative result does not necessarily exclude the proposed model, as there are many possible reasons for the lack of effect, including differential specificity, sensitivity, and affinity for possible binding sites in yeast proteins.
Expression of the five genes also led to greater accumulation of the endocytotic markers lucifer yellow and FM4-64 in yeast. Whereas the basal accumulation of FM4-64 was inhibited by LAT-A, neither the higher rates of FM4-64 uptake nor the enhanced GSH uptake observed in cells expressing the five human genes was affected by LAT-A, indicating that the enhanced GSH uptake associated with expression of the human genes is not mediated by a LAT-A-sensitive endocytotic mechanism. Upon subcellular fractionation, LITAF was found in the P13000 fraction which primarily contains large organelles and the plasma membrane. Indirect immunostaining of LITAF resulted in a staining pattern characteristic of localization to the plasma membrane and intracellular membranes. Mutation of the two PY motifs of LITAF resulted in loss of the observed effects, indicating that this motif plays an essential role in the effects of LITAF expression in yeast.
To date, little is known about the biological functions of the five genes identified in the present study. SLC25A1 is mapped to chromosomal region 22q11, and micro-deletions in this region are associated with DiGeorge syndrome, velo-cardio-facial syndrome and conotruncal anomaly face syndrome [29
]. Although it is not known whether SLC25A1 plays a role in GSH homeostasis, some members of SLC25A family may contribute to mitochondrial GSH uptake. In particular, the dicarboxylate (SLC25A10) and 2-oxoglutarate (SLC25A11) carriers of the inner mitochondrial membrane are believed to catalyze uptake of GSH into the matrix through an exchange mechanism [30
]. The three members of the lysosomal-associated protein transmembrane (LAPTM) family, LAPTM4α, LAPTM4β and LAPTM5 are thought to function as transporters or channels, although their precise function is unknown. When expressed in a drug-sensitive strain of S. cerevisiae, mouse LAPTM4α regulates intracellular compartmentalization of amphipathic solutes and confers cellular resistance or hypersensitivity to a range of drugs [31
]. Both CYYR1 and PMEPA1 have been implicated in human diseases, although their functions are also unknown. CYYR1 is expressed in a broad range of human tissues, and the central cysteine-tyrosine-rich domain is strongly conserved from lower vertebrates to humans [32
]. Alternative splicing and expression of the CYYR1 transcript has been reported in human neuroendocrine tumors [33
]. PMEPA1 was first described as an androgen-induced gene. It is widely expressed in normal and solid malignant tissues, with the highest expression in prostate tissue [34
]. The expression of PMEPA1 is increased in primary and metastasized colon tumors and several other solid tumors [35
], whereas it is decreased in prostate tumor tissues [34
]. In addition, PMEPA1 is also induced by tumor growth factor β [36
]. LITAF is present in most human tissues and its expression can be induced by multiple pathways, including TNFα- and p53-activated pathways [16
]. Elevated mRNA and protein expression of LITAF has been found in intestinal tissues from patients with Crohn's disease and ulcerative colitis [39
]. In addition, mutations of LITAF cause Charcot-Marie-Tooth disease type 1C (CMT1C), an autosomal dominant demyelinating peripheral neuropathy [41
]. Although the etiology of CMT1C is not understood yet, a role of LITAF in modulating protein trafficking and turnover has been proposed to be involved [26
In summary, the present study identifies five human genes that rescue growth of a HGT1-deficient yeast strain, and provides clues into the potential function of these genes. Currently, the functions of the five genes are unknown, although four of them have been implicated in human diseases [35
]. A better understanding of their physiological functions may be expected to clarify the etiology and pathogenesis of these diseases.