Ubiquitinated aldolase B accumulates during starvation-induced lysosomal proteolysis.
We have previously shown that stress-induced protein degradation requires a functional ubiquitin-activating enzyme and the autophagic-lysosomal pathway. In this study, we examined the occurrence of ubiquitin-protein conjugates that form during nutrient starvation. Kidney and liver epithelial cells respond to nutrient stress by enhancing autophagy and protein degradation. We have shown that this degradative response was more dramatic in nondividing cultures. In addition, the onset of autophagy was suppressed by pactamycin, cycloheximide, and puromycin. We observed an accumulation of ubiquitinated proteins coincident with the degradative response to amino acid starvation. The stress-induced protein ubiquitination was not affected by cycloheximide, indicating that protein synthesis was not required. The ubiquitinated proteins were localized to the cytosol and subcellular fractions enriched with autophagosomes and lysosomes. The incorporation of the ubiquitinated proteins into autolysosomes was dramatically reduced by 3-methyladenine, an inhibitor of autophagy. The evidence suggests that ubiquitinated proteins are sequestered by autophagy for degradation. We next set out to identify those primary ubiquitinated proteins at 60 kDa and 68 kDa. Polyclonal antibodies were prepared against these proteins that had been immunopurified from rat liver lysosomes. The antibodies prepared against those 68 kDa proteins also recognized a 40 kDa protein in cytosolic fractions. Internal amino acid sequences obtained from two cyanogen bromide fragments of this 40 kDa protein were shown to be identical to sequences in liver fructose1,6-bisphosphate aldolase B. Anti-Ub68 antibodies recognized purified aldolase A and aldolase B. Conversely, antibodies prepared against aldolase B recognized the 40 kDa aldolase as well as four to five high molecular weight forms, including a 68 kDa protein. Finally, we have shown that the degradation of aldolase B was enhanced during amino acid and serum starvation. This degradation was suppressed by chloroquine and 3-methyladenine, suggesting that aldolase B was being degraded within autolysosomes. We propose that aldolase B is ubiquitinated within the cytosol and then transported into autophagosomes and autolysosomes for degradation during nutrient stress.
Lenk
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Department of Anatomy and Cell Biology, University of Florida College of Medicine, Gainesville, USA.
Susan
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Hickson
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Jasionowski
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Dunn
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D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D000962:Antimalarials / Q000494:pharmacology; D001798:Blood Proteins / Q000378:metabolism / Q000494:pharmacology; D006528:Carcinoma, Hepatocellular; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D002738:Chloroquine / Q000494:pharmacology; D003600:Cytosol / Q000201:enzymology; D005634:Fructose-Bisphosphate Aldolase / Q000235:genetics / Q000378:metabolism*; D007668:Kidney / Q000166:cytology; D008099:Liver / Q000378:metabolism; D008247:Lysosomes / Q000201:enzymology*; D008969:Molecular Sequence Data; D051381:Rats; D013217:Starvation / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D014452:Ubiquitins / Q000378:metabolism*
D000596:Amino Acids; D000962:Antimalarials; D001798:Blood Proteins; D014452:Ubiquitins; C025946:3-methyladenine; D002738:Chloroquine; D005634:Fructose-Bisphosphate Aldolase; D000225:Adenine