Functional analysis of the plasma membrane Alr1 and Alr2 proteins in Yeast Saccharomyces cerevisiae

Marcin Wachek
2009 unpublished
Proteins Alr1 and Alr2 of Saccharomyces cerevisiae encode a very sophisticated system to take up magnesium, one of the most abundant cations, into the cell. In this thesis a series of biochemical and genetic analyses support the functional and structural homology of Alr1p and its close homolog protein Alr2 to the CorA-Mrs2-Alr1 family. Whereas Alr1p acts as the main Mg2+-transporter, Alr2p contributes poorly to Mg2+-uptake. Substitution of a single arginine with a glutamic acid residue in the
more » ... op between the two TM domains greatly improves its activity. Both, Alr1 and Alr2 proteins are shown to form homo- and hetero-oligomers. Furthermore, this assay also showed that the orientation of Alr proteins is consistent with CorA, where both N-terminal and C-terminal ends are exposed to the cell interior. Successive truncations were constructed from N- and C-terminal tails of Alr1p to dissect the role in protein function. The analysis of carboxyl truncations affirmed the importance of this region for Alr1p Mg2+ transport activity and its critical impact on channel formation. In contrast, consecutive shortening of N-terminal domain of Alr1p accounted for striking abnormalities concerning protein stability and subcellular localization. Further analysis using short amino acid intragenic deletions led to the identification of a sequence element of about 50 amino acids in the amino terminal tail essential for protein translocation and stability. Proteins missing this region were blocked in translocation to the plasma membrane and undergo Mg2+ independent degradation, most likely via sorting mechanisms of the ER and subsequential proteasomal decay.
doi:10.25365/thesis.7644 fatcat:rygcq4ejebgmjpnuem4upmhibm