Abstract
Transformation of baker's yeast (Saccharomyces cerevisiae) plays a key role in several experimental techniques, yet the molecular mechanisms underpinning transformation are still unclear. The addition of amino acids to the growth and transformation medium increases transformation efficiency. Here, we show that target of rapamycin complex 1 (TORC1) activated by amino acids enhances transformation via ubiquitin‐mediated endocytosis. We created mutants of the TORC1 pathway, alpha‐arrestins, and eisosome‐related genes. Our results demonstrate that the TORC1‐Npr1‐Art1/Rsp5 pathway regulates yeast transformation. Based on our previous study, activation of this pathway results in up to a 200‐fold increase in transformation efficiency, or greater. Additionally, we suggest DNA may be taken up by domains at the membrane compartment of Can1 (MCC) in the plasma membrane formed by eisosomes. Yeast studies on transformation could be used as a platform to understand the mechanism of DNA uptake in mammalian systems, which is clinically relevant to optimize gene therapy.
Highlights
Yeast transformation is the process by which exogenous DNA is introduced into the cell
We found that mutations of endocytic components resulted in changes in transformation efficiencies supporting the hypothesis that target of rapamycin complex 1 (TORC1) and ubiquitin-mediated endocytosis are keys to yeast transformation
In the evaluation of the transformation efficiency of different budding yeast strains, there was no intergroup comparison, only two groups were compared at a time
Summary
Yeast transformation is the process by which exogenous DNA is introduced into the cell. Foreign DNA is most likely to be engulfed via endocytic membrane invagination, and several mutants involved in endocytosis show low transformation efficiencies (Kawai et al, 2004). Ubiquitination of plasma membrane proteins can serve as an internalization signal for endocytosis (Toret & Drubin, 2006). In this way, the cell can downregulate receptors or transporters via transport to endosomes and lysosomal degradation (Ghaddar et al, 2014). The cell can downregulate receptors or transporters via transport to endosomes and lysosomal degradation (Ghaddar et al, 2014) In yeast, this process is mediated by the Rsp ubiquitin ligase that requires adaptor proteins for recruitment to the specific plasma membrane targets. Several amino acid transporters are internalized by this form of endocytosis, including the general
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