Abstract
Autolysosomes are organelles that sequester and degrade a portion of the cytoplasm during autophagy. Although autophagosomes are short lived compared to other organelles such as mitochondria, plastids, and peroxisomes, many autolysosomes accumulate in tobacco BY-2 cells cultured under sucrose starvation conditions in the presence of a cysteine protease inhibitor. We here describe our methodology for isolating autolysosomes from BY-2 cells by conventional cell fractionation using a Percoll gradient. The autolysosome fraction separates clearly from fractions containing mitochondria and peroxisomes. It contains acid phosphatase, vacuolar H+-ATPase, and protease activity. Electron micrographs show that the fraction contains partially degraded cytoplasm seen in autolysosomes before isolation although an autolysosome structure is only partially preserved.
Keywords:
Acid phosphatase; Autolysosome; Autophagy; Cysteine protease inhibitor; H+-ATPase; Protease; Protein degradation; Sucrose starvation; Vacuole.
Publication types
-
Research Support, Non-U.S. Gov't
MeSH terms
-
Acid Phosphatase / chemistry
-
Acid Phosphatase / isolation & purification
-
Autophagy
-
Biomarkers / chemistry
-
Cell Culture Techniques
-
Cell Fractionation / instrumentation
-
Cell Fractionation / methods*
-
Centrifugation, Density Gradient / instrumentation
-
Centrifugation, Density Gradient / methods*
-
Culture Media / chemistry
-
Cysteine Proteinase Inhibitors / pharmacology
-
Leucine / analogs & derivatives
-
Leucine / pharmacology
-
Lysosomes / chemistry*
-
Lysosomes / drug effects
-
Lysosomes / ultrastructure
-
Mitochondria / chemistry
-
Mitochondria / drug effects
-
Mitochondria / ultrastructure
-
Nicotiana / chemistry
-
Nicotiana / cytology
-
Peptide Hydrolases / chemistry
-
Peptide Hydrolases / isolation & purification
-
Peroxisomes / chemistry
-
Peroxisomes / drug effects
-
Peroxisomes / ultrastructure
-
Plant Cells / chemistry*
-
Plant Cells / drug effects
-
Plant Cells / ultrastructure
-
Plant Proteins / chemistry*
-
Plant Proteins / isolation & purification
-
Proteolysis
-
Sucrose / chemistry
-
Vacuolar Proton-Translocating ATPases / chemistry
-
Vacuolar Proton-Translocating ATPases / isolation & purification
-
Vacuoles / chemistry
-
Vacuoles / drug effects
-
Vacuoles / ultrastructure
Substances
-
Biomarkers
-
Culture Media
-
Cysteine Proteinase Inhibitors
-
Plant Proteins
-
Sucrose
-
N-(N-(3-carboxyoxirane-2-carbonyl)leucyl)isoamylamine
-
Acid Phosphatase
-
Peptide Hydrolases
-
Vacuolar Proton-Translocating ATPases
-
Leucine