Endocytosis is involved in DNA uptake in yeast.
The transfection of mammalian cells by endocytosis is frequently hampered by low efficiency. To identify the bottlenecks, a system that allows the analysis of the intracellular pathway of DNA along the endocytic compartments in the eukaryote Saccharomyces cerevisiae was developed. DNA uptake in yeast cells was achieved by endocytosis when the cells were incubated with episomal DNA in the presence of 34% sucrose and subsequently shifted to a hypotonic medium to induce osmotical lysis of accumulated endocytic intermediates. The compartments of the endocytic pathway after the intersection of the endocytic and the vacuolar sorting pathway are preferred sites of DNA degradation. Either a transport blockade before this point or, even better, the presence of chloroquine, which is known as an adjuvant for transfection in mammalian cells, are required for successful transfection. The transport blockade can be achieved by deleting a GTPase of the endocytic pathway, Ypt51p, or ethanol. Chloroquine affects the compartments of the late endocytic pathway, and no effect is seen on transfection in a strain that is defective for YPT51 and accumulates the DNA in the early endocytic intermediates. To our knowledge, this is the first report on endocytic DNA uptake in S. cerevisiae.
Neukamm
Birgit
B
Department of Microbiology and Genetics, Institute for Biotechnology, Berlin University of Technology, Gustav-Meyer-Allee 25, D-13355 Berlin, Germany.
Stahl
Ulf
U
Lang
Christine
C
D002738:Chloroquine / Q000494:pharmacology; D004247:DNA / Q000378:metabolism*; D004705:Endocytosis / Q000187:drug effects / Q000235:genetics; D017353:Gene Deletion; D009997:Osmotic Pressure; D010957:Plasmids / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D013395:Sucrose / Q000378:metabolism; D013997:Time Factors; D014162:Transfection / Q000379:methods*
D013395:Sucrose; D002738:Chloroquine; D004247:DNA