Identification of Autolysosomes Directly Associated with Proteolysis on the Density Gradients in Isolated Rat Hepatocytes

Abstract

Autophagy-related vacuoles, i.e., autophagosomes (AVi), autolysosomes (AVd) and dense bodies (DB), are intracellular organelles within which macroautophagy and bulk proteolysis set out and progress. Separation of these particles in freshly isolated rat hepatocytes, monitored by beta-hexosaminidase, a lysosomal marker enzyme, was established by density gradient centrifugation. Percoll density gradients were modified and improved by adding free polyvinylpyrrolidone (PVP, 0.75%) to 60% Percoll, which made it possible to separate AVd (buoyant peak, d = 1.090) and DB (dense peak, d = 1.131) effectively. Addition of graded levels of a regulatory amino acid mixture (Reg AA) to hepatocyte incubation not only suppressed proteolysis, but also lead to a shift of vacuolar profiles on the density gradients from the buoyant to the dense region. Alterations in the vacuolar shift and proteolysis were highly proportional over a full range of regulation by Reg AA. Morphometric analysis of autophagic vacuoles by electron microscopy revealed changes in the aggregate volumes of both AVi and AVd by Reg AA, which enabled us to estimate autophagic subpopulation of the buoyant peak on the gradient profile. All the results demonstrate that AVd shifts on the density gradients in proportion to alterations in proteolysis regulated by amino acids, and thus the gradient profile can be used as a measure of macroautophagy; and in addition that AVd actively involved in proteolysis occupies only a part of the buoyant peak on the gradients.

Remodeling the Endoplasmic Reticulum by Poliovirus Infection and by Individual Viral Proteins: an Autophagy-Like Origin for Virus-Induced Vesicles

“…4), providing a potential intermediate in the formation of double-membraned vesicles with cytoplasmic lumen (12,41). Like the poliovirus-induced vesicles studied here, autophagic vacuoles isolated from primary rat hepatocytes migrate very similarly to ER membranes in Percoll gradients, at a lower buoyant density than lysosomes (32). For both autophagic vacuoles and poliovirus-induced vesicles, the apparent exclusion from the ER of protein markers such as calnexin and PDI could be a selective sorting process.…”

Cytosolic LC3 Ratio as a Sensitive Index of Macroautophagy in Isolated Rat Hepatocytes and H4-II-E Cells

“…Normal concentrations (mM) of RegAA, including coregulatory alanine, were as follows: leucine (Leu), 204; tyrosine, 98; proline, 437; methionine, 60; histidine, 92; tryptophan, 93; alanine, 475. 18 For the composition of CAA, refer to reference 18. Subcellular fractionation from hepatocytes.…”

“…In order to keep organelles and membranes as intact as possible, a tightly fitting Dounce homogenizer (7 ml size, Wheaton Science Products, NJ, USA) was employed for 120 strokes on ice, resulting in a mild and mechanical homogenization. 18 Differential centrifugation was performed as previously described, 19 and each fraction was washed twice to remove contamination from other fractions. From total homogenates (TH), which consisted of the nuclear (N), mitochondrial-lysosomal (ML), microsomal pellet (P), and soluble cytosolic (S) fractions, samples were obtained as 700 x g precipitates, 17,500 x g precipitates, 100,000 x g precipitates and supernatants, respectively.…”

Amino Acids and Insulin Control Autophagic Proteolysis through Different Signaling Pathways in Relation to mTOR in Isolated Rat Hepatocytes

“…The amino acids employed were those that have been shown to exert direct effects on autophagic proteolysis, called regulatory amino acids, and were added as multiples of the normal plasma concentrations as shown in the figure legends. The normal concentrations (1ϫ) of the regulatory amino acids including coregulatory Ala (RegAA) were as follows (M): Leu, 204; Tyr, 98; Pro, 437; Met, 60; His, 92; Trp, 93; Ala, 475 (33).…”

“…The supernatants were stored at Ϫ20°C until analysis. Val was derivatized with dansyl chloride, using L-norvaline as an internal standard, and separated by reverse phase-high performance liquid chromatography using a Supelcosil LC-18 column (4.6 ϫ 150 mm) as described previously (33). As another proteolytic measurement without cycloheximide, the rates of protein synthesis and net Val release were measured simultaneously, and the proteolytic rate was calculated from both rates (36).…”