Novel system for monitoring autophagy in the yeast Saccharomyces cerevisiae.
The yeast S. cerevisiae imports cytosolic components into the vacuole non-selectively by autophagy and degrades them by vacuolar hydrolases under nutrient starvation conditions. We developed a novel system for monitoring autophagy by constructing cells in which modified vacuolar alkaline phosphatase is expressed as an inactive precursor form in the cytosol. Under starvation conditions, the processing of the precursor to the mature form and phosphatase activity appeared gradually, and the mature form was located in the vacuole. Disruption of APG1, an essential gene for autophagy, resulted in no processing or phosphatase activity. These results indicate that the precursor form in the cytosol is transferred to the vacuole by autophagy and converted to the active form by vacuolar proteinases. Thus, autophagy could be determined easily and accurately by measuring the phosphatase activity.
Noda
T
T
Department of Biology, College of Arts and Sciences, University of Tokyo, Japan.
Matsuura
A
A
Wada
Y
Y
Ohsumi
Y
Y
D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001343:Autophagy; D001483:Base Sequence; D002451:Cell Compartmentation; D017931:DNA Primers / Q000737:chemistry; D005656:Fungal Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D014617:Vacuoles / Q000201:enzymology
D017931:DNA Primers; D005656:Fungal Proteins; D008565:Membrane Proteins; D000469:Alkaline Phosphatase