Defects in intracellular trafficking and endocytic/vacuolar acidification determine the efficiency of endocytotic DNA uptake in yeast.
The yeast Saccharomyces cerevisiae is a standard model system to study endocytosis. Here we describe the examination of a representative subset of deletion mutants to identify and locate steps in endocytic transport, endosomal/lysosomal acidification and in intracellular transport of hydrolases in non-viral transfection processes. When transport in late endocytosis is inhibited, transfection efficiency is significantly enhanced. Similarly, transfection efficiency is enhanced when the pH-value of the endosomal/vacuolar system is modified. Transfection efficiency is furthermore elevated when the N+/K+ transport in the endosomal system is disturbed. Finally, we observe enhanced transfection efficiency in mutants disturbed in the CVT/autophagy pathway and in hydrolase transport to the vacuole. In summary, non-viral transfection efficiency can be significantly increased by either (i) inhibiting the transport of endocytosed material before it enters the vacuole, or (ii) inducing a non-natural pH-value of the endosomal/vacuolar system, or (iii) slowing down degradative processes by inhibiting vacuolar hydrolases or the transport between Golgi and late endosome/vacuole.
Riechers
Sean-Patrick
SP
Department of Microbiology and Genetics, Institute for Biotechnology, Technische Universität Berlin, Gustav-Meyer-Allee 25, D-13355 Berlin, Germany.
Stahl
Ulf
U
Lang
Christine
C
D000143:Acids; D001692:Biological Transport; D004271:DNA, Fungal / Q000378:metabolism*; D004705:Endocytosis; D006863:Hydrogen-Ion Concentration; D009154:Mutation / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D019076:Transgenes / Q000235:genetics; D014617:Vacuoles / Q000378:metabolism*
D000143:Acids; D004271:DNA, Fungal