ASNA1 is a well-conserved ATPase involved in a wide range of functions, including cisplatin resistance, growth control, insulin secretion and targeting of tail-anchored (TA) proteins to membranes. It is a positive regulator of insulin secretion both in the roundworm Caenorhabditis elegans and in humans. Insulin secretion and downstream insulin/IGF signalling (IIS) stands at the heart of many human pathologies, such as diabetes, Alzheimer’s disease and cancer. A better understanding of IIS may therefore prove vital for treatment and cure of these diseases. This thesis aims to further investigate the function of asna-1, and to identify new regulators of IIS based on the asna-1 phenotype in C. elegans.
Worms lacking ASNA-1 arrest growth in the first larval stage, L1, with reduced insulin secretion. The L1 arrest represents the strongest of the IIS phenotypes in worms. Most regulators of the insulin pathway have been identified in screens for other IIS phenotypes, influencing lifespan or the dauer diapause. Therefore, new regulators could be found by screening for genes which, when inactivated, cause an asna-1-like L1 arrest. Using bioinformatic approaches, a set of 143 putative asna-1 interactors were identified, based on their predicted or confirmed interaction with asna-1 in various organisms. Depletion of the Golgi SNARE homologue YKT-6 or the mitochondrial translocase homologue TOMM-40 caused asna-1-like larval arrests. Using several criteria, including genetic suppression by daf-16/Foxo, it was established that YKT-6 and TOMM-40 are positive regulators of IIS. Both proteins were also required for normal DAF-28/insulin secretion.
Further investigation of TOMM-40 identified it as a ubiquitously expressed mitochondrial translocase in C. elegans: It localized to mitochondrial membranes and was required for importing a tagged mitochondrial reporter across mitochondrial membranes. Depletion of TOMM-40 caused a collapse of the proton gradient across the inner mitochondrial membrane and triggered the mitochondrial unfolded protein response (UPR). Worms with defective mitochondria failed to grow normally in presence of food, but this growth defect was suppressed by daf-16(mgDf50). In addition, tomm-40(RNAi) led to DAF-16/FOXO activation, an effect that was suppressed by over expression of DAF-28/insulin. Taken together, these findings support a model whereby signals of food availability are conveyed through respiring mitochondria to promote DAF-28/insulin secretion, which in turn promotes growth.
Biochemical studies have identified ASNA-1 as a chaperone that targets a subset of newly synthesized TA proteins to a receptor at the endoplasmic reticulum (ER) membrane. However, these findings have not been tested in vivo in a metazoan model. A reporter-based system to analyse TA protein targeting into the ER in live animals using confocal microscopy was set up. A model asna-1-dependent TA protein, Y38F2AR.9/SEC-61β, required functional ASNA-1 for correct targeting to the ER. Conversely, a model asna-1-independent TA protein, CYTB5.1/cytochrome B5, did not. This phenotype was shared with the predicted asna-1 receptor homologue, wrb-1. Consistently, WRB-1 was found to localize to the ER. However, other wrb-1 mutant phenotypes only partially overlap with those of asna-1 mutants, suggesting that ASNA-1 is either partially independent of WRB-1 for TA protein targeting or that ASNA-1 has additional functions besides its role in TA protein targeting.
Confocal microscopy also indicated that the ER morphology was aberrant in asna-1 and wrb-1 mutants. ER UPR was elevated in the asna-1 mutants, as indicated by the upregulation of an hsp-4/BiP reporter. Transmission and immuno-electron microscopy of these mutants revealed a swollen ER lumen, which is another hallmark of ER stress. High levels of autophagy in asna-1 animals and the presence of ER-containing autophagosomes in both asna-1 and wrb-1 mutants indicated a stress-induced remodelling of the ER membrane in these two mutants. In addition, both mutants had normal mitochondrial morphology, but showed severe effects on Golgi compartment morphology. Hypothetically, all these phenotypes could be due to defects in the signal recognition particle (SRP) pathway. This is because Y38F2AR.9/SEC-61β is both a TA protein and a component of the SEC-61 translocon. However, both Golgi and ER morphology was normal in Y38F2AR.9/sec-61β(tm1986) mutant animals, suggesting that the organellar defects seen in asna-1 and wrb-1 were due to a TA protein-dependent mechanism rather than an SRP-dependent mechanism. In addition, asna-1 mutants displayed numerous protein aggregates, consistent with a proposed role for ASNA-1 in shielding aggregation-prone TA protein membrane anchors from the hydrophilic environment of the cytosol.
In conclusion, YKT-6 and TOMM-40 are positive regulators of IIS and DAF-28/insulin secretion, implicating roles for Golgi and mitochondria in IIS. DAF-28 is a metabolically regulated insulin in C. elegans, since its secretion depends on active mitochondria. Mutants for asna-1 and its predicted receptor wrb-1 show severe defects in ER and Golgi morphology. These defects may occur because TA protein targeting in asna-1 and wrb-1 mutants is defective, which is also demonstrated here in the first analysis of this process in live animals.