Characterization of GET Protein Distribution in S.cerevisiae Under Different Physiological Conditions – From Light Microscopy to Ultrastructural Analysis

Tail-anchored (TA) proteins destined to enter the secretory pathway are post-translationally inserted into the endoplasmic reticulum (ER) membrane by the GET (guided entry of TA proteins) pathway. The cytosolic ATPase Get3 recognizes the single transmembrane domain in the C-terminal tail of TA proteins and mediates their delivery to the ER membrane-bound receptors Get1 and Get2 followed by insertion of the TA protein into the membrane.

We analyzed Get3 localization under different physiological conditions and found that it relocates from the cytosol and the ER membrane to punctate structures upon glucose-starvation in a Get4/5-dependent manner. Similarly, a Get3 ATP hydrolysis mutant accumulates in puncta. Upon glucose addition, Get3 redistributes to the cytosol again indicating that Get3 localization is a dynamic and adaptive mechanism affected by the energy status of the cell.

Additionally, in absence of its receptors Get1/2, Get3 is also detected in Get4-dependent puncta. Co-localization using various marker proteins could not yet classify Get3 puncta suggesting they may represent a so far uncharacterized supra-molecular assembly in the cell. Employing transmission electron microscopy we characterized the ultra-structural features of Get3 puncta under different conditions. We hypothesize that Get3 plays a crucial role in the adaptation of TA protein biogenesis to energy metabolism and to conditions of inefficient protein insertion into the ER membrane.