Abstract
Early-onset torsion dystonia is a severe, life-long disease that leads to loss of motor control and involuntary muscle contractions. While the molecular etiology of the disease is not fully understood, a mutation in an AAA+ ATPase, torsinA, has been linked to disease onset. Previous work on torsinA has shown that it localizes to the endoplasmic reticulum, where there is evidence that it plays roles in protein trafficking, and potentially also protein folding. Given the high level of evolutionary conservation among proteins involved in these processes, the ability of human such proteins to function effectively in yeast, as well as the previous successes achieved in examining other proteins involved in complex human diseases in yeast, we hypothesized that Saccharomyces cerevisiae might represent a useful model system for studying torsinA function and the effects of its mutants. Since torsinA is proposed to function in protein homeostasis, we tested cells for their ability to respond to various stressors, using a fluorescent reporter to measure the unfolded protein response, as well as their rate of protein secretion. TorsinA did not impact these processes, even after co-expression of its recently identified interacting partner, printor. In light of these findings, we propose that yeast may lack an additional cofactor necessary for torsinA function or proteins required for essential post-translational modifications of torsinA. Alternatively, torsinA may not function in endoplasmic reticulum protein homeostasis. The strains and assays we describe may provide useful tools for identifying and investigating these possibilities and are freely available.
Highlights
A glutamic acid deletion (DE) in torsinA is associated with earlyonset torsion dystonia, a devastating autosomal dominant neurofunctional disease that impacts patients as children or young adults and has no cure [1]
We replaced the endogenous endoplasmic reticulum (ER) localization sequence of torsinA [4] with the localization sequence of the yeast ER-resident protein, Kar2p (Figure 1A) [31]. In this construct, only the portion of torsinA that has been shown previously to be cleaved following ER localization was replaced [3,5], leaving all functional domains of the final protein intact. This caused torsinA to localize to the ER, as visualized by C-terminal green fluorescent protein (GFP) tagging of torsinA (Figure 1B)
For all experiments other than microscopy, the GFP tag was replaced with an HDEL (ER retention) motif to ensure that any torsinA mistakenly trafficked to the Golgi apparatus would be returned to the ER [32]
Summary
A glutamic acid deletion (DE) in torsinA is associated with earlyonset torsion dystonia, a devastating autosomal dominant neurofunctional disease that impacts patients as children or young adults and has no cure [1]. C-terminal to this is a hydrophobic region that allows torsinA to interact with membranes [5]. The remainder of the protein encompasses an AAA+ ATPase domain, which includes the site of the dystonia-associated mutation [6]. While AAA+ ATPase domains have been ascribed diverse functions, many are involved in protein remodeling [7]. Yeast have AAA+ ATPases that participate in protein folding, such as HSP104 [8], but none are homologs of torsinA. There is precedent for successfully studying AAA+ ATPases in heterologous environments, as HSP104 has been shown to function in neurons, which lack an HSP104 homolog [9]
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