Abstract
The autophagosomes were identified in the viable cycloheximide (CHX)-treated cells which had an incapacitated translational process and thus disabled synthesis of endoplasmic reticulum (ER)-derived vesicular transporters. They were found devoid of the proteins transported from ER to cell organelles, were unable to fuse with ER, Golgi or mitochondria, and displayed affinity with lysosomes. The analysis of autophagosomes, derived from the CHX cell organelles, revealed that their lipid composition resemble that of the maternal organelle. Thus, the ER-derived autophagosomes were marked with the presence of phosphatidylinositol (PI), Golgi-derived vesicles contained sphingomyelin (SM) and glycosphingolipids (GLL), and the mitochondria-derived autophagosomes contained phosphatidylglycerol (PG) and cardiolipin (CL). The incubation of the vesicles with intact lysosomes afforded their and the lysosome membrane lipids degradation. The analysis of the products derived from incubation of lysosomes and autophagosomes with radiolabeled SM, in the presence and the absence of TritonX100, allowed us to conclude that during autophagosome degradation the lysosomal enzymes are not released to cytosol, and that only lysosomes contain the enzymes degrading membrane lipids. In summary, our findings allowed us to authenticate the vesicles generated in the CHX-treated cells as organelle-specific autophagosomes and to determine that complete cycle of cell restitution and debridement includes intralysosomal degradation of the lysosomal membrane engulfing the autophagosomes vesicles.
Highlights
In theory, an autophagy, the degradation of the dysfunctional portions of cell membrane and its organelles, and its synchronization with biomembranes repair and replacement is crucial to maintain the state of equilibrium in the cell size, specificity and function
In the experiments dedicated to the vesicles synthesis with translation active Cell Cytosol (CC) or RNase or CHX treated CC, the preparations of the organelles were rinsed with phosphate buffered saline (PBS), or 0.5 M NaCl or urea-PBS, in order to remove the associated residual cytosolic proteins that otherwise would remain on their membranes
The subcellular fractions representing endoplasmic reticulum (ER), Golgi, and mitochondria were subjected to the incubation with the transport active CHX CC to produce organelle specific vesicles which were tested for their ability to associate and fuse with cell organelles and their lipid composition characterized
Summary
An autophagy, the degradation of the dysfunctional portions of cell membrane and its organelles, and its synchronization with biomembranes repair and replacement is crucial to maintain the state of equilibrium in the cell size, specificity and function. Knocking out gene technique is the most popular way to probe a protein’s destination and function, but such manipulation can create unnoticeable yet relevant consequence in the transport, by generating central change that destroys destination marker of the transporter [4,5,6,7,8,9]. The results of the studies embracing genetic manipulation and point mutation techniques are assessed under the conditions that minimize function of membrane-specific lipids or reduce their involvement to mere hydrophobic background for the protein folding
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