De novo autophagic vacuole formation in hepatocytes permeabilized by Staphylococcus aureus alpha-toxin. Inhibition by nonhydrolyzable GTP analogs.
The role of GTP-binding proteins in autophagic vacuole formation was investigated in isolated rat hepatocytes permeabilized by alpha-toxin from Staphylococcus aureus, an agent which creates stable plasma membrane channels allowing exchange of small (< or = 1000 Da) molecules. Vacuole formation was monitored from the uptake of 125I-tyramine-cellobiitol (125ITC) into osmotically sensitive vacuoles isolated on colloidal silica density gradients. Separation was based on an established observation that autophagic vacuoles are retained in a heavy midgradient band when samples are layered, but are selectively shifted to dense fractions when they are previously dispersed in the gradient material. The vacuolar uptake of 125ITC was concentration-dependent and required exogenous ATP: 94% was directly mediated by sequestration; 6% was acquired by fluid-phase endocytosis as monitored by [carboxyl-14C]dextran-carboxyl. Although the amino acid control of proteolysis was lost, addition of the nonhydrolyzable GTP analog GTP gamma S (as well as GMP-PNP) decreased fractional rates of direct vacuolar 125ITC uptake and long-lived proteolysis by similar amounts (1.02-1.03% h-1), substantiating the notion that the effects were the direct result of autophagic inhibition. These and associated findings, supported by quantitative electron microscopy, indicate the presence of ongoing macro- and microautophagy in alpha-toxin-permeabilized cells and suggest that one or more GTP-binding proteins is required in macroautophagic vacuole formation.
Kadowaki
M
M
Department of Cellular and Molecular Physiology, College of Medicine, Pennsylvania State University, Hershey 17033.
Venerando
R
R
Miotto
G
G
Mortimore
G E
GE
D000818:Animals; D001329:Autolysis; D001427:Bacterial Toxins / Q000037:antagonists & inhibitors / Q000633:toxicity*; D002462:Cell Membrane / Q000187:drug effects / Q000648:ultrastructure; D002463:Cell Membrane Permeability / Q000187:drug effects*; D002478:Cells, Cultured; D004187:Disaccharides / Q000378:metabolism; D005098:Exotoxins / Q000633:toxicity*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology*; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D006460:Hemolysin Proteins / Q000633:toxicity*; D007457:Iodine Radioisotopes; D008099:Liver / Q000187:drug effects / Q000648:ultrastructure*; D008297:Male; D008854:Microscopy, Electron; D051381:Rats; D011917:Rats, Inbred Lew; D013211:Staphylococcus aureus; D013873:Thionucleotides / Q000494:pharmacology; D014439:Tyramine / Q000031:analogs & derivatives / Q000378:metabolism; D014617:Vacuoles / Q000187:drug effects* / Q000648:ultrastructure; D014633:Valine / Q000032:analysis
D001427:Bacterial Toxins; D004187:Disaccharides; D005098:Exotoxins; D006460:Hemolysin Proteins; D007457:Iodine Radioisotopes; D013873:Thionucleotides; C085840:iodotyramine-cellobiitol; C021091:staphylococcal alpha-toxin; D006153:Guanosine Diphosphate; D006165:Guanylyl Imidodiphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C023427:guanosine 5'-O-(2-thiodiphosphate); D006160:Guanosine Triphosphate; D014633:Valine; D014439:Tyramine