Energy dependence of different steps in the autophagic-lysosomal pathway.
The energy dependence of the autophagic-lysosomal pathway was investigated in isolated rat hepatocytes, using electroinjected [14C]lactose as an autophagy probe and atractyloside to alter intracellular ATP levels. Since autophagocytosed lactose is hydrolyzed in lysosomes, several steps in the pathway could be analyzed. The following observations were made. 1) The overall autophagic degradation of electroinjected [14C]lactose was strongly energy-dependent. More than 85% inhibition was obtained when the ATP content decreased from the control value of 10 mumol/g dry weight to 4 mumol/g dry weight. 2) The initial step, i.e. the autophagic sequestration of [14C]lactose, measured in the presence of vinblastine to prevent transfer of lactose to lysosomes, was as sensitive to small changes in ATP as was the overall lactose degradation. 3) The steady state level of sequestered [14C]lactose remained constant as ATP decreased from 10 to 4 mumol/g dry weight, indicating that the sequestration step and some postsequestrational process were inhibited to a similar extent by ATP depletion. 4) The final step in the pathway, intralysosomal hydrolysis, was measured by allowing [14C]lactose to preaccumulate intralysosomally in the presence of the reversible lysosome inhibitor propylamine. Following propylamine removal and inhibition of further sequestration by 3-methyladenine, ATP-dependent hydrolysis of the intralysosomal [14C]lactose could be demonstrated. However, this hydrolysis step was not as sensitive to small changes in ATP as was the sequestration step or the overall autophagic lactose degradation. Control of the autophagic-lysosomal pathway in response to energy deprivation would therefore not seem to occur at the lysosomal level, but may be exerted both at the sequestration step and at a postsequestrational, prelysosomal step.
Plomp
P J
PJ
Laboratory of Biochemistry, University of Amsterdam, The Netherlands.
Gordon
P B
PB
Meijer
A J
AJ
Høyvik
H
H
Seglen
P O
PO
D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001278:Atractyloside / Q000494:pharmacology; D001343:Autophagy / Q000187:drug effects; D002451:Cell Compartmentation / Q000187:drug effects; D004734:Energy Metabolism; D007785:Lactose / Q000378:metabolism; D008247:Lysosomes / Q000502:physiology*; D008297:Male; D010587:Phagocytosis / Q000187:drug effects; D011437:Propylamines / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D013395:Sucrose / Q000378:metabolism; D014747:Vinblastine / Q000494:pharmacology
D011437:Propylamines; D001278:Atractyloside; C025946:3-methyladenine; D013395:Sucrose; D014747:Vinblastine; D000255:Adenosine Triphosphate; D007785:Lactose; D000225:Adenine