Kinetics, dynamics and localization of basic amino acid transporters in Saccharomyces cerevisiae

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Kinetics, dynamics and localization of basic amino acid transporters in Saccharomyces cerevisiae
PhD ceremony:F. Bianchi, MSc
When:April 29, 2016 
Promotor:prof. dr. B. (Bert) Poolman
Where:Academy building RUG
Faculty:Mathematics and Natural Sciences


Amino acid permeases (AAPs) in the plasma membrane (PM) of Saccharomyces cerevisiae are responsible for the uptake and regulation of the cellular levels of amino acids. The focus of my thesis is on the AAP’s responsible for the uptake of basic amino acids. Curiously these AAPs have been reported to function unidirectionally, something that is not easily understood on the basis of how this class of transport proteins is thought to work. Furthermore, for unknown reasons, AAPs very similar in sequence localize in different compartments of the plasma membrane.

To address the kinetics of transport we purified the Lyp1 protein and studied its properties in synthetic membranes. To address the membrane biogenesis of the transporters, we tagged the Lyp1 and Can1 proteins, predicted to reside in different membrane compartments, with a fluorescent protein and used super-resolution optical microscopy and single-molecule tracking to determine their traffic and localization in the cell.

We find that contrary to published data, Lyp1 functions bidirectionally. We conclude that previous observation were mistakenly interpreted as unidirectional transport, because the affinity constants for out-to-in and in-to-out transport differ by several orders of magnitude and basic amino acids are partially stored in the vacuole.

We also report on a conserved signature sequence in the C-terminus of AAPs, which modulates the function and localization of AAPs along the PM, Finally, we explain the localization of Lyp1 and Can1 in the plasma membrane to the distinct sites where the proteins are inserted in the membrane, their relatively short lifetime and the slow diffusion away from the site of insertion. Overall, our work provides a comprehensive understanding of the energetics, kinetics and biogenesis of plasma membrane transporters in yeast.


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