Publicação
Endocytic trafficking and ubiquitylation of plasma membrane transporters in yeast
| Resumo: | Plasma membrane proteins play a crucial role on cellular physiology, including receptor signaling and uptake of nutrients, neurotransmission and removal of toxic compounds. Endocytosis allows the remodelling of the membrane protein repertoire. Highly evolutionarily conserved post-translational modifications (notably ubiquitylation) were proven to be critical for trafficking, endocytosis and membrane protein degradation. The impairment of the regulated trafficking of membrane proteins such as receptors, transporters and channels is closely associated to several human diseases ethology including hypertension, cardiovascular diseases, diabetes, neurodegenerative diseases and cancer. In fungi, the endocytic pathway of nutrient transporters requires arrestin-like adaptors or simply α-arrestins, which contains PY motifs to directly interact with the Nedd4-like Rsp5 E3 enzyme, being then adaptors for the ubiquitylation system. These α-arrestins are also phospho-regulated by signaling pathways, being a bridge between endocytosis and signaling. The lactate transporter Jen1 from Saccharomyces cerevisiae has been used as a eukaryotic nutrient transporter model. In this thesis, a review on the recent literature regarding carboxylic acid plasma membrane transporters, in S. cerevisiae, was performed, approaching many aspects of cellular physiology, such as function, structure, regulation and evolution. Previous works established that the ubiquitylation and endocytosis of Jen1 lactate transporter, in response to glucose, requires Rod1/Art4 α-arrestin, which is regulated by AMPK/Snf1 kinase and Reg1/PP1 phosphatase. In this thesis, a novel mechanism, requiring the α-arrestin Bul1, was discovered to be involved in the ubiquitylation, endocytosis and vacuolar degradation of Jen1, in response to cycloheximide, prolonged growth in lactate (alkalinization) or rapamycin. This process requires, in some cases, the signaling pathway TORC1 complex and some of its effectors (Tco89 subunit, Tor1 protein, Sit4 phosphatase and Npr1 kinase). Importantly, the activity of Jen1 transporter seems to be required for α-arrestin Bul1 activity, according to the signal. The role of the cytosolic N- and C-terminal regions of the Jen1 transporter of S. cerevisiae was also studied in respect to subcellular trafficking, transport activity and turnover, Rational design and functional analysis of specific Jen1 truncations and chimeric transporters were carried out, based on Jen1 and two other transporters of S. cerevisiae, Gap1 and Fur4, or on the purine transporter UapA from Aspergillus nidulans. Our results show that both cytosolic terminals are important for Jen1 function and regulation. The Nterminal region proved critical for subcellular localization and essential for transport activity and glucose- or alkalinization-elicited endocytosis. The direct involvement of the N-terminal tail in Rod1-dependent, glucose-elicited, endocytic turnover was strongly supported by its ability to confer glucose-elicited endocytosis to UapA, a transporter that is normally stably expressed in S. cerevisiae. The C-terminal region of Jen1 was also shown to be essential for transport activity and critical for trafficking and signal-elicited endocytosis. The results presented herein point to the idea that cytosolic terminals of Jen1 interact and, together, determine its endocytic turnover. |
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| Autores principais: | Rocha, Gabriel Azevedo Carreira Talaia da |
| Assunto: | Ciências Naturais::Ciências Biológicas |
| Ano: | 2019 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso restrito |
| Instituição associada: | Universidade do Minho |
| Idioma: | inglês |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | Plasma membrane proteins play a crucial role on cellular physiology, including receptor signaling and uptake of nutrients, neurotransmission and removal of toxic compounds. Endocytosis allows the remodelling of the membrane protein repertoire. Highly evolutionarily conserved post-translational modifications (notably ubiquitylation) were proven to be critical for trafficking, endocytosis and membrane protein degradation. The impairment of the regulated trafficking of membrane proteins such as receptors, transporters and channels is closely associated to several human diseases ethology including hypertension, cardiovascular diseases, diabetes, neurodegenerative diseases and cancer. In fungi, the endocytic pathway of nutrient transporters requires arrestin-like adaptors or simply α-arrestins, which contains PY motifs to directly interact with the Nedd4-like Rsp5 E3 enzyme, being then adaptors for the ubiquitylation system. These α-arrestins are also phospho-regulated by signaling pathways, being a bridge between endocytosis and signaling. The lactate transporter Jen1 from Saccharomyces cerevisiae has been used as a eukaryotic nutrient transporter model. In this thesis, a review on the recent literature regarding carboxylic acid plasma membrane transporters, in S. cerevisiae, was performed, approaching many aspects of cellular physiology, such as function, structure, regulation and evolution. Previous works established that the ubiquitylation and endocytosis of Jen1 lactate transporter, in response to glucose, requires Rod1/Art4 α-arrestin, which is regulated by AMPK/Snf1 kinase and Reg1/PP1 phosphatase. In this thesis, a novel mechanism, requiring the α-arrestin Bul1, was discovered to be involved in the ubiquitylation, endocytosis and vacuolar degradation of Jen1, in response to cycloheximide, prolonged growth in lactate (alkalinization) or rapamycin. This process requires, in some cases, the signaling pathway TORC1 complex and some of its effectors (Tco89 subunit, Tor1 protein, Sit4 phosphatase and Npr1 kinase). Importantly, the activity of Jen1 transporter seems to be required for α-arrestin Bul1 activity, according to the signal. The role of the cytosolic N- and C-terminal regions of the Jen1 transporter of S. cerevisiae was also studied in respect to subcellular trafficking, transport activity and turnover, Rational design and functional analysis of specific Jen1 truncations and chimeric transporters were carried out, based on Jen1 and two other transporters of S. cerevisiae, Gap1 and Fur4, or on the purine transporter UapA from Aspergillus nidulans. Our results show that both cytosolic terminals are important for Jen1 function and regulation. The Nterminal region proved critical for subcellular localization and essential for transport activity and glucose- or alkalinization-elicited endocytosis. The direct involvement of the N-terminal tail in Rod1-dependent, glucose-elicited, endocytic turnover was strongly supported by its ability to confer glucose-elicited endocytosis to UapA, a transporter that is normally stably expressed in S. cerevisiae. The C-terminal region of Jen1 was also shown to be essential for transport activity and critical for trafficking and signal-elicited endocytosis. The results presented herein point to the idea that cytosolic terminals of Jen1 interact and, together, determine its endocytic turnover. |
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