Ubiquitin-activating enzyme, E1, is associated with maturation of autophagic vacuoles.

The ubiquitin-activating enzyme, E1, is required for initiating a multi-step pathway for the covalent linkage of ubiquitin to target proteins. A CHO cell line containing a mutant thermolabile E1, ts20, has been shown to be defective in stress-induced degradation of proteins at restrictive temperature (Gropper et al., 1991. J. Biol. Chem. 266:3602-3610). Parental E36 cells responded to restrictive temperature by stimulating lysosome-mediated protein degradation twofold. Such a response was not observed in ts20 cells. The absence of accelerated degradation in these cells at 39.5 degrees C was accompanied by an accumulation of autolysosomes. The fractional volume of these degradative autophagic vacuoles was at least sixfold greater than that observed for either E36 cells at 30.5 degrees or 39.5 degrees C, or ts20 cells at 30.5 degrees C. These vacuoles were acidic and contained both acid phosphatase and cathepsin L, but, unlike the autolysosomes observed in E36 cells, ubiquitin-conjugated proteins were conspicuously absent. Combined, our results suggest that in ts20 cells, which are unable to generate ubiquitin-protein conjugates due to heat inactivation of E1, the formation and maturation of autophagosomes into autolysosomes is normal, but the conversion of autolysosomes into residual bodies is disrupted.

Regulation of protein secretion by crinophagy in perfused rat liver.

We examined the secretion of three serum proteins, albumin (RSA), alpha 2 mu-globulin (alpha 2 mu G), and transferrin (Trf), in the isolated perfused liver. Within 4 h of perfusion, only 20 to 35% of previously synthesized proteins were secreted by the liver into the recirculating medium. Low temperature inhibited the secretion of alpha 2 mu G and Trf, but not RSA. The amount of RSA secreted by the liver increased twofold in the presence of leupeptin, a proteinase inhibitor, or primaquine, a weak base capable of neutralizing acidic compartments. Neither drug affected Trf secretion, while the release of alpha 2 mu G was enhanced threefold by primaquine treatment. Only 55 to 70% of the total amount of these serum proteins present in the liver at the onset of perfusion could be accounted for after 4 h of perfusion. Our evidence suggests that these losses are due to protein degradation. The degradation of RSA and alpha 2 mu G was inhibited at 15 degrees C and by both leupeptin and primaquine. Contrary, RSA degradation was not altered when livers were perfused at 20 degrees C. Morphological techniques combined with immunological probes were utilized to identify possible intracellular sites of RSA degradation. RSA and cathepsin L were colocalized to large vacuoles found near the cell periphery. Entry of RSA into these vacuoles occurred at 20 degrees C but not at 15 degrees C. Our results using perfused rat livers suggest that as much as 40% of hepatic serum proteins are degraded via fusion of secretory vesicles with lysosomes (e.g., crinophagy).

Angiotensin II regulation of plasminogen activator inhibitor-1 gene expression in neurons of normotensive and spontaneously hypertensive rat brains.

Neuronal cells in primary culture from the hypothalamus-brain stem areas of normotensive [Wistar-Kyoto (WKY)] and spontaneously hypertensive (SH) rat brains have been used in the present study to investigate an interaction between the brain renin-angiotensin II system and the plasminogen activator system. This is an attempt to further our understanding of the role of brain Ang II in the control of neuronal development and differentiation through its regulation of the extracellular matrix. Ang II caused a 10-fold stimulation of plasminogen activator inhibitor-1 (PAI-1) messenger RNA (mRNA) in WKY rat brain neuronal cultures. The stimulation was mediated by the AT1 receptor subtype and was accompanied by an increase in PAI-1 gene transcription and the synthesis of cellular PAI-1 protein. The stimulation involved activation of protein kinase C, and alterations in the intracellular Ca2+ pool caused a significant inhibition of Ang II stimulation of PAI mRNA. Ang II stimulation of PAI-1 mRNA succeeded its action on c-fos mRNA and was attenuated by c-fos antisense oligonucleotide. Although PAI-1 gene expression was also stimulated by Ang II in neuronal cultures of SH rat brain, two differences between WKY and SH rat brain neurons were observed: 1) the level of Ang II stimulation in SH rat neurons was 50% of that in WKY rat neurons; and 2) Ang II stimulation of c-fos was 2.4-fold higher in SH neurons than in WKY neurons, but c-fos antisense oligonucleotide did not attenuate the stimulatory action of Ang II on PAI-1 mRNA in SH neurons. These observations suggest that the changes in the Ang II-mediated signaling pathways and/or the regulatory region(s) of the PAI-1 gene may contribute to the differential actions of Ang II in WKY and SH rat brain neurons.

Ultrastructure of feeding in the karyorelictean ciliate Tracheloraphis examined by scanning electron microscopy.

The karyorelictean ciliate Tracheloraphis is considered to be among the most primitive of the extant ciliates based on both nuclear and somatic characters. These organisms lack the elaborate oral ciliation present in most ciliates. Their mode of ingestion is a type of phagocytosis through a non-ciliated region, the glabrous stripe, which runs the length of the cell. This type of ingestion is reminiscent of feeding in amoebae and some flagellate groups. It is possible that ciliate oral structures evolved within the karyorelictean ciliates from an ancestor resembling Tracheloraphis. We studied the ingestion process in a Tracheloraphis species from the Chesapeake Bay using scanning electron microscopy. The results indicate that the anterior terminus of the organism is not involved in the actual ingestion process, only the glabrous stripe. There is some interaction between the food particle and the surface of the stripe, possibly mediated by a substance secreted by the underlying extrusomes. The somatic cilia do not appear to be involved. The stripe invaginates at the ingestion site engulfing the particle. The cell becomes greatly distended at this site, but neither the anterior nor posterior terminus is affected.

Cytoskeletal elements are required for the formation and maturation of autophagic vacuoles.

We evaluated the role of cytoskeletal elements in the degradation of endogenous proteins via autophagy using biochemical and morphological techniques. In the absence of exogenous amino acids, degradation of endogenous proteins was enhanced in cultured normal rat kidney cells. This enhanced degradative state was accompanied by a 4-fold increase in the occurrence of autophagic vacuoles. In the presence of drugs that induce the depolymerization of microfilaments (cytochalasins B and D) or microtubules (nocodazole), protein degradation was not enhanced in nutrient-deprived cells. Although these drugs had similar inhibitory effects on the protein degradation, their effect on autophagy differed. Cytochalasins B and D interfered with the formation of the autophagosome. In cells treated with these drugs, the fractional volume represented by autophagic vacuoles was not substantially increased despite nutrient depletion. On the contrary, nocodazole appeared to have no effect on the formation of autophagosomes. Instead, this drug suppressed the delivery of hydrolytic enzymes, thereby resulting in an accumulation of acidic autophagic vacuoles containing undegraded cellular components.

Effects of streptozotocin-induced diabetes on rough endoplasmic reticulum and lysosomes of rat liver.

In the absence of amino acids and insulin, ribosome-free regions of the rough endoplasmic reticulum (RER) invaginate to form an autophagosome, which matures into an autolysosome (W. A. Dunn, Jr., J. Cell Biol. 110: 1923-1933, 1990). In this study, biochemical and morphological methods were used to examine the structure and integrity of the RER and the lysosome-vacuolar system in livers of untreated (normal serum insulin) and streptozotocin (STZ)-treated (depressed serum insulin) fed and fasted rats. Degradation of endogenous proteins was increased by 70% in STZ-treated animals. Proteolysis was further enhanced when these animals were deprived of food for 24 h. These alterations in protein turnover were accompanied by increases in the fractional volume of autophagic vacuoles and in the hepatic amounts of three lysosomal hydrolases. These effects of STZ were prevented on administration of insulin. In addition, there was an insulin-dependent 50% loss of RER surface area in livers from STZ-treated rats. This loss of structural RER was accompanied by comparable decreases in the cellular amounts of two RER membrane proteins and one luminal protein, suggesting that the RER was degraded as a unit. Additional losses of RER were observed when STZ-treated rats were fasted. Furthermore, the hepatic amounts of two serum proteins decreased, suggesting the functional capacity of the RER was reduced. Combined, the data suggest that in STZ-induced diabetes the losses in RER are related to enhanced autophagy.

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.