Abstract
The degradation of intra- and extracellular proteins is essential in all cell types and mediated by two systems, the ubiquitin-proteasome system (UPS) and the autophagy-lysosome pathway. This study investigates the changes in autophagosomal and lysosomal proteomes upon inhibition of proteasomes by bortezomib (BTZ) or MG132. We find an increased abundance of more than 50 proteins in lysosomes of cells in which the proteasome is inhibited. Among those are dihydrofolate reductase (DHFR), β-Catenin and 3-hydroxy-3-methylglutaryl-coenzym-A (HMGCoA)-reductase. Because these proteins are known to be degraded by the proteasome they seem to be compensatorily delivered to the autophagosomal pathway when the proteasome is inactivated. Surprisingly, most of the proteins which show increased amounts in the lysosomes of BTZ or MG132 treated cells are proteasomal subunits. Thus an inactivated, non-functional proteasome is delivered to the autophagic pathway. Native gel electrophoresis shows that the proteasome reaches the lysosome intact and not disassembled. Adaptor proteins, which target proteasomes to autophagy, have been described in Arabidopsis, Saccharomyces and upon starvation in mammalians. However, in cell lines deficient of these proteins or their mammalian orthologues, respectively, the transfer of proteasomes to the lysosome is not impaired. Obviously, these proteins do not play a role as autophagy adaptor proteins in mammalian cells. We can also show that chaperone-mediated autophagy (CMA) does not participate in the proteasome delivery to the lysosomes. In autophagy-related (ATG)-5 and ATG7 deficient cells the delivery of inactivated proteasomes to the autophagic pathway was only partially blocked, indicating the existence of at least two different pathways by which inactivated proteasomes can be delivered to the lysosome in mammalian cells.
Highlights
Two distinct mechanisms, the autophagy-lysosome pathway and the ubiquitin-proteasome system (UPS), mediate protein degradation within the cell
Analysis of Proteomic Changes in Lysosome-enriched Fractions After Proteasome Inhibition—To determine changes in the lysosomal protein composition caused by an impairment of the ubiquitin-proteasome system, a SILAC-based mass spectrometric analysis [18] of lysosomes from cells treated with either MG132 or BTZ was performed
HEK293 cells were grown in complete DMEM containing either light or heavy amino acids for six passages before incubating them with superparamagnetic particles for 24 h followed by a 36 h chase to allow complete transport of magnetic particles to the lysosome
Summary
The autophagy-lysosome pathway and the UPS, mediate protein degradation within the cell. The generation of autophagosomes starts by the formation and growth of a double-layered isolation membrane enabled by specialized autophagy proteins like the microtubule-associated protein light chain 3 (LC3) in the cytoplasm, engulfing cargo that is to be degraded [3]. An in-depth investigation to specify the detailed mechanism revealed that inactivated proteasomes are most likely degraded by macroautophagy and not by chaperone-mediated autophagy but do still reach lysosomes when either ATG5 or ATG7 are deficient This process is neither dependent on Toll-interacting protein (TOLLIP); the adaptor protein for proteaphagy in yeast, nor SQSTM1, and is not accompanied by an increased association of any known macroautophagy adaptor protein to inactivated proteasomes
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