Palmitoylation is a post-translational modification that has been implicated in the control of multiple proteins, including ion channels. S-Palmitoylation is a lipophilic modification that involves the attachment of palmitate through a thioester linkage to a cysteine residue in a target protein. By increasing the hydrophobicity of the target region, palmitoylation can promote membrane targeting. Here, palmitoylation is shown to play an important role in regulating large conductance calcium- and voltage- activated (BK) potassium channels. The STREX splice variant of the BK channel contains a 58 amino acid insert at the splice site C2 within the intracellular C-terminal RCK1-RCK2 linker that confers increased calcium sensitivity to the channel and determines PKA inhibition of channel activity. The cysteine rich STREX domain was predicted to be palmitoylated, and using an imaging assay STREX was shown to act as a membrane targeting domain through palmitoylation of a di-cysteine motif (C645:C645). A membrane potential assay and electrophysiological analysis demonstrates that palmitoylation at the C645:C646 site in STREX is important in mediating the increased calcium sensitive properties inherent to the STREX channel. Palmitoylation is also shown to modulate PKA channel inhibition. The stability of palmitoylation can often be reliant on the local environment within the protein. Generally in most proteins; lipidated regions, basic domains or transmembrane domains are found adjacent to a palmitoylation site. In STREX, a polybasic domain composed of 11 basic residues just upstream from the C645:C646 palmitoylation site, functions to control the palmitoylation status of the STREX insert. A site directed mutagenesis approach to disrupt the polybasic domain revealed an important role in controlling membrane targeting of the STREX C-terminus, mediating the increased calcium sensitivity inherent to STREX channels and controlling the palmitoylation status of the C645:C646 palmitoylation site using multiple techniques involving electrophysiology, fluorescent imaging and biochemical assays. Further to this, using imaging to examine the membrane association of fluorescently tagged C-terminal proteins, phosphorylation is shown to function as a physiological electrostatic switch to regulate the polybasic region in controlling palmitoylation of the STREX insert. Finally, an additional palmitoylation site that is constitutively expressed in all BK channels was identified to be located in the S0-S1 linker (C53:C54:C56). Mutation of the C53:C54:C56 palmitoylation site in the S0-S1 linker was shown to abolish all palmitoylation in BK channels that did not contain the STREX insert. Palmitoylation allows the S0-S1 linker to associate with the plasma membrane however the mutated de-palmitoylated channels did not affect channel conductance or the calcium/voltage sensitivity of the channel. Palmitoylation of the S0-S1 linker was shown to be a critical determinant of cell surface expression of BK channels, as steady state surface expression levels were reduced by ~55% in the C53:C54:C56 mutant. STREX channels that could not be palmitoylated in the S0-S1 linker also showed decreased surface expression even through STREX insert palmitoylation was unaffected. Palmitoylation is rapidly emerging as an important post-translational mechanism to control ion channel behaviour. This work reveals that palmitoylation of the BK channel can control channel function of the STREX splice variant channel and can regulate cell surface expression in all other channel variants. Palmitoylation appears to be functionally independent at these two distinct sites expressed within the same channel protein.

Ras is a central player in signal transduction that mediates cellular proliferation and differentiation. Recent evidence has shown that lipid and non-lipid modified domains participate in Ras traffic and that plasma membrane association is mediated by vectorial vesicular transport from the endomembrane system. ERp57, an ER chaperone, has been shown to specifically bind farnesylated Hras but not non-farnesylated Hras. The objective of this study was to determine if ERp57 participates in Ras trafficking and function. First, the effect of ERp57 knock down by siRNA technology on Hras function was studied; there was a reduction in ERp57 cellular levels that led to a decrease of active ras. Second, specific anti-ERp57 antibodies were delivered into 3T3 cells expressing GFP-ras chimeras to observe the effect on intracellular trafficking. Anti-ERp57 antibodies blocked Hras plasma membrane localization but not Kras suggesting that ERp57 may be involved in Hras intracellular trafficking and function.

Rho-GTPases are essential signalling proteins which regulate a multitude of central cellular processes that are vital for organisms to thrive and adapt to changing environments. Many regulatory networks involving Rho proteins have first been elucidated in the model yeast Saccharomyces cerevisiae, in which Rho5 emerges as a central hub connecting different signalling pathways, such as the responses to cell wall stress, high medium osmolarity, and oxidative stress. In this work, the rapid translocation of Rho5 to mitochondria as reaction to oxidants and glucose starvation was thoroughly investigated. The studies on structure-function relationships was focussed on the C-terminal region of the Rho5 which in other Rho-type GTPases determines their spatio-temporal distribution and contributes to their physiological function. The C-terminal end of these GTPases is considered to be a hypervariable region (HPR) that consists of a polybasic region (PBR) and its preceding amino acid residues, followed by the CAAX motif which becomes prenylated at its cysteine residue. These motifs are conserved in the yeast Rho5 where the PBR contains a serine residue as a putative phosphorylation target. Moreover, Rho5 of S. cerevisiae is characterized by an extension preceding the PBR that comprises 98 amino acid residues. While substitutions of the serine residue within the PBR for either phosphomimetic or non-phosphorylatable residues indicate that it is of minor physiological importance, deletion analyses of the yeast-specific extension showed that it is required for proper localization of Rho5 to the plasma membrane. As expected, substitution of the cysteine residue within the CAAX motif also prevented proper plasma membrane localization, accompanied by a loss of function both with respect to oxidative stress response and glucose starvation. Results from studies employing a trapping-device of GFP-Rho5 to the mitochondrial surface indicate that the GTPase needs to be activated at the plasma membrane by its dimeric GDP/GTP exchange factor (GEF) which is composed of Dck1 and Lmo1, in response to stress conditions. The trimeric DLR complex is then capable of rapidly translocate to mitochondria and fulfil its functions at the organelle. This view was supported by the finding that a constitutively active Rho5 variant restored function when trapped to mitochondria. Interestingly, Rho5 requires the dimeric GEF for the translocation process under oxidative stress while Dck1 and Lmo1 can reach the mitochondria independent from each other. Finally, the human Rho5 homolog Rac1 cannot complement the defects of a rho5 deletion and does not show a proper intracellular distribution, unless its C-terminal end is equipped with the yeast-specific extension. Taken together, the results of this thesis contributed to a better understanding of the structure-function relationships of Rho5 and its human homolog Rac1.

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