Catching targeting factors involved in co-translational import into mitochondria

Abstract

Cells within multicellular organisms have different compartments called organelles. One of these organelles is the mitochondrium, which provides the cell with the energy required to perform its functions. Most of the proteins within the mitochondria are brought there after they are synthesized by a molecular machine called the ribosome. This synthesis process is called translation. However, sometimes the proteins are also brought to the mitochondria while being translated, which is called co-translational targeting. Until now, co-translational targeting to the mitochondria has not been studied that well. In the yeast S.cerevisiae, it has been found that the nascent polypeptide-associated complex (NAC) interacts with actively translating ribosomes called ribosome nascent-chain complexes (RNCs). It is unknown if NAC has the same functions in mammals as in yeast. Moreover, there is hardly any structural information on the interaction between NAC and RNCs. This thesis aimed to find targeting factors like NAC in mammalian and yeast systems. For this aim, a system that can translate the proteins is needed. This system is called an in vitro translation system. Therefore, a yeast in vitro translation system was established and optimized in the lab. To generate the RNCs, model proteins were chosen. The DNA of these model proteins was supplemented with a sequence that halts the translation by the ribosome to allow for the generation of RNCs. In addition, it contains a sequence that allows for the purification of the RNCs. The model proteins were then translated in the yeast in vitro and in a previously established mammalian in vitro translation system called rabbit reticulocyte lysate (RRL). The RNCs and empty ribosomes were isolated from the in vitro translation mixtures and imaged using negative stain electron microscopy (EM), which Is a technique that allow us to look at small molecules that we can not see with our eye. With this method, we could determine the sample homogeneity. The RRL RNCs were, but the yeast RNCs were not homogenous. Moreover, purification of the RNCs was attempted; however, this was unsuccessful. Before we can find mitochondrial targeting factors, the purification of the RNCs needs to be optimized. When this is done, cryo-single particle analysis EM (cryo-SPA-EM) can be performed, which is a microscopy technique that allows us to look at molecules with higher detail than with negative stain EM. However, this also requires further optimization. Lastly, the newly established yeast in vitro translation system can be used for other applications such as research in other fields.