Tryptophan is a relatively rare amino acid whose influx is strictly controlled to meet cellular demands. The yeast Saccharomyces cerevisiae has two tryptophan permeases, namely Tat1 (low-affinity type) and Tat2 (high-affinity type). These permeases are differentially regulated through ubiquitination based on inducible conditions and dependence on arrestin-related trafficking adaptors, although the physiological significance of their degradation remain unclear. Here, we demonstrated that Tat2 was rapidly degraded in an Rsp5-Bul1-dependent manner upon the addition of tryptophan, phenylalanine, or tyrosine, whereas Tat1 was unaffected. The expression of the ubiquitination-deficient variant Tat2^5K>R led to a reduction in cell yield at 4 μg/mL tryptophan, suggesting the occurrence of an uncontrolled, excessive consumption of tryptophan at low tryptophan concentrations. Eisosomes are membrane furrows that are thought to be storage compartments for some nutrient permeases. Tryptophan addition caused rapid Tat2 dissociation from eisosomes, whereas Tat1 distribution was unaffected. The 5 K > R mutation had no marked effect on Tat2 dissociation, suggesting that dissociation is independent of ubiquitination. Interestingly, the D74R mutation, which was created within the N-terminal acidic patch, stabilized Tat2 while reducing the degree of partitioning into eisosomes. Moreover, the hyperactive I285V mutation in Tat2, which increases V_max/K_m for tryptophan import by 2-fold, reduced the degree of segregation into eisosomes. Our findings illustrate the coordinated activity of Tat1 and Tat2 in the regulation of tryptophan transport at various tryptophan concentrations and suggest the positive role of substrates in inducing a conformational transition in Tat2, resulting in its dissociation from eisosomes and subsequent ubiquitination-dependent degradation.

• PMID: 35031272
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• PubMed

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Journal Article | Research Support, Non-U.S. Gov't

Authors

Ishii R, Fukui A, Sakihama Y, Kitsukawa S, Futami A, Mochizuki T, Nagano M, Toshima J, Abe F

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