Magnesium, the second most abundant cation in the cell, is involved in a diverse range of biochemical activities. This project focuses on the mechanism of magnesium import into the cell through the action of Alr1p. Alr1p resides in the plasma membrane of yeast and belongs to the CorA-Alr1p-Mrs2p family of magnesium channels. Potential regulators of CorA were found through genetic screening and yeast two-hybrid screens have pulled out interactors of Alr1p. Interactors that influence Alr1p and its conformation will, with very high probability, also change the channel’s ability for magnesium import. Membrane proteins are not easily amenable to traditional yeast two-hybrid screens due to their hydrophobic nature. The goal of this thesis is to identify interactors of Alr1p using iMYTH, a modified yeast two-hybrid method. Of the eighteen Alr1p interactors identified, Vma3p and Vma11p, which are both subunits of the V-ATPase, showed the most promise for further Alr1p interaction characterizations.

Magnesium, the second most abundant cation in the cell, is involved in a diverse range of biochemical activities. This project focuses on the mechanism of magnesium import into the cell through the action of Alr1p. Alr1p resides in the plasma membrane of yeast and belongs to the CorA-Alr1p-Mrs2p family of magnesium channels. Potential regulators of CorA were found through genetic screening and yeast two-hybrid screens have pulled out interactors of Alr1p. Interactors that influence Alr1p and its conformation will, with very high probability, also change the channel’s ability for magnesium import. Membrane proteins are not easily amenable to traditional yeast two-hybrid screens due to their hydrophobic nature. The goal of this thesis is to identify interactors of Alr1p using iMYTH, a modified yeast two-hybrid method. Of the eighteen Alr1p interactors identified, Vma3p and Vma11p, which are both subunits of the V-ATPase, showed the most promise for further Alr1p interaction characterizations.

Paramecium tetraurelia is a ciliated single cell used as a model organism for the study of ciliopathies. Ciliopathies are mammalian diseases involving the dysfunction of cilia, including cilia maintenance, construction, and signaling. P. tetraurelia and its cilia provides an excellent non-canonical system for the investigation and elucidation of proteins important for the structure, maintenance and function of cilia and ciliary beating. We utilize features of this cell such as its 1000's of cilia and highly organized and patterned cell surface to observe changes in swimming behavior or disruptions in the ordered cell surface which are not feasible in mammalian cells. Here, we present research on three proteins in Paramecium, two of which are homologs to human ciliopathy genes. Using combinations of epitope-tagging, RNA interference (RNAi), immunofluorescence, immunoprecipitations, LC-MS/MS analysis and electrophysiology, we have attempted to elucidate the location, function, and potential interacting partners of these three proteins. The first protein, meckelin (MKS3), is a contributing factor in Meckel-Gruber syndrome, among other ciliopathies. Using epitope tagging, we identified the location of the Mks3 protein above each basal body. Depletion of MKS3 using RNAi leads to global loss of cilia, a severe disruption in the surface organization and a mislocalization of basal bodies out of the anterior-posterior axis of the cell. We show that depletion of Mks3 leads to abnormal backward swimming in ionic stimuli and depleted secretion of trichocysts. Based on our data, we propose two functions for Mks3 in P. tetraurelia. The first function is a transition zone component important for proper regulation of ciliary protein content, consistent with MKS3 function in other organisms. The depletion of MKS3 led to global ciliary loss, but also an imbalance in the ciliary ion channels that was different from the loss of cilia due to interference with intraflagellar transport as observed in cells depleted of IFT88. The second novel role for MKS3 is as a transient connection to the kinetodesmal fiber which is important for basal body guidance when daughter basal bodies migrate away from the mother basal body before cell division. We also examine the contribution of the non-selective cation channel Polycystin-2 (Pkd2) in Paramecium to Mg2+ permeability and Mg2+-induced behavior. When mutated in humans, Pkd2 leads to 15% of the cases of Autosomal Dominant Polycystic Kidney Disease (ADPKD). When PKD2 is depleted using RNAi in Paramecium, cells show short backward swimming in Mg2+ solutions, a resistance to heavy metal paralysis, and depleted membrane permeability to Mg2+. The channel-like protein XntA which is unique to Paramecium and Tetrahymena, is also important for these phenotypes. Therefore, we utilized the Paramecium XntA1 mutant in our studies, which lacks Mg2+-induced behavior. We demonstrate that both Pkd2 and XntA are present in the cell membrane and in the cilia. Co-IP assays show that the IP of XntA-myc co-IPs the Pkd2-FLAG channel, but not vice versa, possibly because of an occluded FLAG epitope due to protein interactions. To tease apart the contributions of Pkd2 in the cilia and the cell membrane, electrophysiology was used to measure membrane potential of ciliated and deciliated cells. Depletion of BBS8 eliminates Pkd2 in the cilia, allowing us to examine Pkd2 activity restricted to the cell membrane of ciliated cells. Depletion of Pkd2 or XntA decreases membrane permeability to Mg2+. When Pkd2 was restricted to the cell membrane via BBS8 depletion, the membrane permeability to Mg2+ increased, much like over-expressing the Pkd2 protein. Depletion of Pkd2, especially in the deciliated XntA1 mutant, leads to a dramatic decrease in Mg2+ membrane permeability. Based on these data, we propose that Pkd2 is the Mg2+ channel in Paramecium and XntA is not a channel, but is perhaps important for stabilizing Pkd2 in membrane microdomains. We have uncovered novel function roles for the proteins mentioned here, leading to a broader understanding of their function. These studies also highlight to usefulness and importance of the model organism Paramecium tetraurelia to the study of human ciliopathy genes.