Abstract
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.
Highlights
The role of GTP-bindingproteins in autophagic vacu- GTP-binding proteins havebeen implicated in an increasing ole formation was investigated in isolated rat hepato- number of membrane events suchas endosome fusion, vesicle cytes permeabilizedby a-toxinfrom Staphylococcus au- formation by the endoplasmic reticulum, the Golgilsecretory reus, an agent which creates stable plasma membrane pathway, exocytosis, a n d glucose transporter translocation('7
Separation was basedon an establishedobservation that autophagic vacuoles are retained in a heavy midgradient band when sampalrees layered, but are selectively shiftedto dense hctions when theyare previously dispersedin the gradient materiTalh. e vacu
Duced poresare sufficientlylarge to allow exchangeof nucleotides across the plasma membrane (131, the possibilitywas down of proteinpreviously sequestered in autophagicvacuconsidered that proteolysis would be adversely affected by a oles),rates from 30 to 90(or 120min, not shown) wecroenstant diminished energy supply
Summary
(Received for publication, June 16, 1993, and in revised form, September 7 , 1993). Motoni KadowakiS, Rina Venerandog, GiovannMi iotto[8], and Glenn E. The abbreviations used are: GTPyS, guanosine 5'-0-(3-thiotriphosphate); GDPPS,guanyl-5'-yl thiophosphate; GMP-PNP, guanyl-5'-yl imidodiphosphate; ImITC, 'mI-tyramine-cellobiitol; M + L, mitochondrial-lysosomal fraction; ~ hexos ~ d a s e , ~ - a c e ~ l - ~ - D - gluco ~ dase; A%, ~ t i ~m a~~ a ~u t onp h v~tacuole ( autop ~ g ~ o m e ) ; AVd, de~adativddigestivemacroautophagic vacuole(autolysosome); ER, endoplasmic reticulum In experiments where unlabeled M + L from intact liver was mixed with labeled M + L from incubated cells, a vertical rotor of larger capacity (SS-90)was employed In these instances, 1.0-ml samples containing 28.4 mg of protein were used, and centrifugation was carried out at 18,000rpm for 150min. The significance of differences betweenmeans was evaluated by the Student’s t test; values o f p greater than 0.05 were considered not significant
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
