Determination of Four Sequential Stages during Microautophagy in Vitro
2004; Elsevier BV; Volume: 279; Issue: 11 Linguagem: Inglês
10.1074/jbc.m307905200
ISSN1083-351X
AutoresJoachim B. Kunz, Heinz Schwarz, Andreas Mayer,
Tópico(s)Cellular transport and secretion
ResumoMicroautophagy is the transfer of cytosolic components into the lysosome by direct invagination of the lysosomal membrane and subsequent budding of vesicles into the lysosomal lumen. This process is topologically equivalent to membrane invagination during multivesicular body formation and to the budding of enveloped viruses. Vacuoles are lysosomal compartments of yeasts. Vacuolar membrane invagination can be reconstituted in vitro with purified yeast vacuoles, serving as a model system for budding of vesicles into the lumen of an organelle. Using this in vitro system, we defined different reaction states. We identified inhibitors of microautophagy in vitro and used them as tools for kinetic analysis. This allowed us to characterize four biochemically distinguishable steps of the reaction. We propose that these correspond to sequential stages of vacuole invagination and vesicle scission. Formation of vacuolar invaginations was slow and temperature-dependent, whereas the final scission of the vesicle from a preformed invagination was fast and proceeded even on ice. Our observations suggest that the formation of invaginations rather than the scission of vesicles is the rate-limiting step of the overall reaction. Microautophagy is the transfer of cytosolic components into the lysosome by direct invagination of the lysosomal membrane and subsequent budding of vesicles into the lysosomal lumen. This process is topologically equivalent to membrane invagination during multivesicular body formation and to the budding of enveloped viruses. Vacuoles are lysosomal compartments of yeasts. Vacuolar membrane invagination can be reconstituted in vitro with purified yeast vacuoles, serving as a model system for budding of vesicles into the lumen of an organelle. Using this in vitro system, we defined different reaction states. We identified inhibitors of microautophagy in vitro and used them as tools for kinetic analysis. This allowed us to characterize four biochemically distinguishable steps of the reaction. We propose that these correspond to sequential stages of vacuole invagination and vesicle scission. Formation of vacuolar invaginations was slow and temperature-dependent, whereas the final scission of the vesicle from a preformed invagination was fast and proceeded even on ice. Our observations suggest that the formation of invaginations rather than the scission of vesicles is the rate-limiting step of the overall reaction. The lysosome is a major lytic compartment in eukaryotic cells (1Knop M. Schiffer H.H. Rupp S. Wolf D.H. Curr. Opin. Cell Biol. 1993; 5: 990-996Crossref PubMed Scopus (83) Google Scholar). Substrates are delivered to its lumen by several pathways: via endocytosis, via direct transport across the lysosomal membrane, and via autophagy (2Dunn Jr., W.A. Trends Cell Biol. 1994; 4: 139-143Abstract Full Text PDF PubMed Scopus (444) Google Scholar, 3Khalfan W.A. Klionsky D.J. Curr. Opin. Cell Biol. 2002; 14: 468-475Crossref PubMed Scopus (58) Google Scholar, 4Noda T. Suzuki K. Ohsumi Y. Trends Cell Biol. 2002; 12: 231-235Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). Autophagy, defined as nonselective uptake and degradation of cytoplasm in the lysosome, can occur either as macro- or as microautophagy. In macroautophagy, nascent autophagosomes engulf parts of the cytoplasm and subsequently fuse with the lysosome. During microautophagy, vesicles bud into the lysosomal lumen by direct invagination of the boundary membrane, resulting in degradation of both cytoplasmic components and lysosomal membrane. Microautophagy can lead to the degradation of soluble components or to the selective uptake of entire organelles, as exemplified by peroxisome degradation (micropexophagy). In order to distinguish these two forms, which we presume to follow different mechanisms, we refer to the microautophagic uptake of soluble components as type I and to that of organelles as type II. Vacuoles are the lysosomal compartment of the yeast Saccharomyces cerevisiae. Tubular invaginations leading to type I microautophagy have been observed both in living yeast cells and in isolated vacuoles (5Müller O. Sattler T. Flötenmeyer M. Schwarz H. Plattner H. Mayer A. J. Cell Biol. 2000; 151: 519-528Crossref PubMed Scopus (141) Google Scholar). These membrane tubules are induced upon starvation. They are dynamic and often branched structures showing a sharp kink of the vacuolar membrane at the site of invagination. Since the lumen of the tubules is continuous with the cytoplasm, budding of vesicles from the tip of the tubules results in unilamellar autophagic bodies containing cytosol, destined for degradation by vacuolar hydrolases. Because of a lateral heterogeneity along the autophagic tubes, with a high density of transmembrane particles at the base and a smooth zone at the tip where budding occurs, the autophagic bodies reaching the vacuolar lumen are largely devoid of transmembrane particles. The process of type I vacuole invagination and vesicle formation can be reconstituted in vitro using purified yeast vacuoles (6Sattler T. Mayer A. J. Cell Biol. 2000; 151: 529-538Crossref PubMed Scopus (88) Google Scholar). Solute uptake can be quantified using luciferase as a reporter enzyme. Luciferase uptake depends on ATP, salt concentration, temperature, and cytosol. Cytosolic extracts prepared from starved cells stimulate luciferase uptake by isolated vacuoles with an activity 2-fold higher than that of extracts from nonstarved cells. Cytosolic extracts from starved atg mutants support the in vitro reaction to a similar extent as extracts from nonstarved wild type cells. This observation suggests that Atg proteins may be involved in regulation of type I microautophagy but not in the uptake reaction itself. Microautophagy does not depend on known factors for vacuole fusion and vesicle trafficking, such as Sec17p/α-SNAP; Sec18p/NSF; or the SNAREs Vam3p, Vam7p, or Nyv1p. Scission of the invaginated membrane hence occurs via a mechanism distinct from homotypic vacuole fusion. Pioneering genetic screens have identified many genes required for macroautophagy (3Khalfan W.A. Klionsky D.J. Curr. Opin. Cell Biol. 2002; 14: 468-475Crossref PubMed Scopus (58) Google Scholar, 4Noda T. Suzuki K. Ohsumi Y. Trends Cell Biol. 2002; 12: 231-235Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 7Tsukada M. Ohsumi Y. FEBS Lett. 1993; 333: 169-174Crossref PubMed Scopus (1427) Google Scholar, 8Thumm M. Egner R. Koch B. Schlumpberger M. Straub M. Veenhuis M. Wolf D.H. FEBS Lett. 1994; 349: 275-280Crossref PubMed Scopus (483) Google Scholar, 9Barth H. Thumm M. Gene (Amst.). 2001; 274: 151-156Crossref PubMed Scopus (30) Google Scholar, 10Epple U.D. Suriapranata I. Eskelinen E.L. Thumm M. J. Bacteriol. 2001; 183: 5942-5945Crossref PubMed Scopus (147) Google Scholar, 11Shintani T. Suzuki K. Kamada Y. Noda T. Ohsumi Y. J. Biol. Chem. 2001; 276: 30452-30460Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 12Wang C.W. Kim J. Huang W.P. Ableiovich H. Stromhaug P.E. Dunn Jr., W.A. Klionsky D.J. J. Biol. Chem. 2001; 276: 30442-30451Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 13Hutchins M.U. Klionsky D.J. J. Biol. Chem. 2001; 276: 20491-20498Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 14Komatsu M. Tanida I. Ueno T. Ohsumi M. Ohsumi Y. Kominami E. J. Biol. Chem. 2001; 276: 9846-9854Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 15Kim J. Huang W.P. Klionsky D.J. J. Cell Biol. 2001; 152: 51-64Crossref PubMed Scopus (188) Google Scholar, 16Ichimura Y. Kirisako T. Takao T. Satomi Y. Shimonishi Y. Ishihara N. Mizushima N. Tanida I. Kominami E. Ohsumi M. Noda T. Ohsumi Y. Nature. 2000; 408: 488-492Crossref PubMed Scopus (1542) Google Scholar, 17Abeliovich H. Dunn Jr., W.A. Kim J. Klionsky D.J. J. Cell Biol. 2000; 151: 1025-1034Crossref PubMed Scopus (236) Google Scholar, 18Suriaprinata I. Epple U.D. Bernreuther D. Bredschneider M. Sovarasteanu K. Thumm M. J. Cell Sci. 2000; 113: 4025-4033Crossref PubMed Google Scholar, 19Kamada Y. Funakoshi T. Shintani T. Nagano K. Ohsumi M. Ohsumi Y. J. Cell Biol. 2000; 150: 1507-1513Crossref PubMed Scopus (914) Google Scholar, 20Scott S.V. Nice 3rd., D.C. Nau J.J. Weisman L.S. Kamada Y. Keizer-Gunnink I. Funakoshi T. Veenhuis M. Ohsumi Y. Klionsky D.J. J. Biol. Chem. 2000; 275: 25840-25849Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 21Lang T. Reiche S. Straub M. Bredschneider M. Thumm M. J. Bacteriol. 2000; 182: 2125-2133Crossref PubMed Scopus (81) Google Scholar, 22Harding T.M. Hefner-Gravink A. Thumm M. Klionsky D.J. J. Biol. Chem. 1996; 271: 17621-17624Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, 23Mizushima N. Noda T. Yoshinori T. Tanaka Y. Ishii T. George M.D. Klionsky D.J. Ohsumi M. Ohsumi Y. Nature. 1998; 395: 395-398Crossref PubMed Scopus (1297) Google Scholar, 24Lang T. Schaeffeler E. Bernreuther D. Bredschneider M. Wolf D.H. Thumm M. EMBO J. 1998; 17: 3597-3607Crossref PubMed Scopus (230) Google Scholar, 25Meiling-Wesse K. Barth H. Voss C. Barmark G. Muren E. Ronne H. Thumm M. FEBS Lett. 2002; 530: 174-180Crossref PubMed Scopus (24) Google Scholar, 26Wang C.W. Stromhaug P.E. Shima J. Klionsky D.J. J. Biol. Chem. 2002; 277: 47917-47927Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 27Meiling-Wesse K. Barth H. Thumm M. FEBS Lett. 2002; 526: 71-76Crossref PubMed Scopus (50) Google Scholar, 28Kuma A. Mizushima N. Ishihara N. Ohsumi Y. J. Biol. Chem. 2002; 277: 18619-18625Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar, 29Barth H. Meiling-Wesse K. Epple U.D. Thumm M. FEBS Lett. 2001; 508: 23-28Crossref PubMed Scopus (92) Google Scholar, 30Titorenko V.I. Keizer I. Harder W. Veenhuis M. J. Bacteriol. 1995; 177: 357-363Crossref PubMed Scopus (93) Google Scholar, 31Mukaiyama H. Oku M. Baba M. Samizo T. Hammond A.T. Glick B.S. Kato N. Sakai Y. Genes Cells. 2002; 7: 75-90Crossref PubMed Scopus (101) Google Scholar) and for autophagic peroxisome degradation (30Titorenko V.I. Keizer I. Harder W. Veenhuis M. J. Bacteriol. 1995; 177: 357-363Crossref PubMed Scopus (93) Google Scholar, 31Mukaiyama H. Oku M. Baba M. Samizo T. Hammond A.T. Glick B.S. Kato N. Sakai Y. Genes Cells. 2002; 7: 75-90Crossref PubMed Scopus (101) Google Scholar, 32Sakai Y. Koller A. Rangell L.K. Keller G.A. Subramani S. J. Cell Biol. 1998; 141: 625-636Crossref PubMed Scopus (197) Google Scholar, 33Yuan W. Tuttle D.L. Shi Y.J. Ralph G.S. Dunn Jr., W.A. J. Cell Sci. 1997; 110: 1935-1945Crossref PubMed Google Scholar). They have shed light on the mechanisms underlying these important protein degradation pathways. However, mutants selectively defective in microautophagy of soluble cytosolic components have not been identified so far. No method for the selective quantitation of type I microautophagy in living cells is available other than morphological observation of vacuolar membrane invaginations (5Müller O. Sattler T. Flötenmeyer M. Schwarz H. Plattner H. Mayer A. J. Cell Biol. 2000; 151: 519-528Crossref PubMed Scopus (141) Google Scholar, 6Sattler T. Mayer A. J. Cell Biol. 2000; 151: 529-538Crossref PubMed Scopus (88) Google Scholar). In order to further characterize microautophagic vesicle formation, we used a cell-free system that reconstitutes type I vacuole invagination, and we performed time course experiments to dissect the reaction into four steps. Chemicals—Nystatin, amphotericin, FCCP, and valinomycin were purchased from Sigma. Nocodazol, colchicine, rapamycin, K252a, and aristolochic acid were purchased from Alexis (Gruenberg, Germany). GTPγS 1The abbreviations used are: GTPγS, guanosine 5′-3-O-(thio)triphosphate; FCCP, carbonylcyanide-p-trifluoromethoxyphenylhydrazone; PIPES, 1,4-piperazinediethanesulfonic acid; PS, PIPES/sorbitol; GDPβS, guanyl-5′-yl thiophosphate. was purchased from Roche Applied Science. All other reagents were analytical grade. Drugs were suspended as 50× or 100× stock solution in Me2SO and stored at –20 °C. Nystatin stock solutions were renewed after 1 week of storage. Yeast culture, cytosol preparation, and vacuole preparation were performed as described previously (6Sattler T. Mayer A. J. Cell Biol. 2000; 151: 529-538Crossref PubMed Scopus (88) Google Scholar). For storage of vacuoles, protease inhibitors (0.2 mm pefabloc SC, 0.2 μg/ml leupeptin, 1 mm o-phenanthroline, 1 μg/ml pepstatin A) and glycerol (10% (w/v) from a 50% stock) were added to a fresh vacuole suspension. The suspension was frozen as little nuggets in liquid nitrogen and stored at –80 °C for a maximum of 3 weeks. In Vitro Microautophagy Assay—A standard reaction had a volume of 50 μl and was composed of vacuoles (0.2 mg/ml, strain DBY5734 (6Sattler T. Mayer A. J. Cell Biol. 2000; 151: 529-538Crossref PubMed Scopus (88) Google Scholar), either freshly prepared or thawed from a –80 °C stock), 3 mg/ml cytosol from starved K91–1A cells (6Sattler T. Mayer A. J. Cell Biol. 2000; 151: 529-538Crossref PubMed Scopus (88) Google Scholar), 105 mm KCl, 7 mm MgCl2, 2 mm ATP, 80 mm disodium creatine phosphate, 175 units/ml creatine kinase, 17 μg/ml luciferase, 100 μm dithiothreitol, 0.1 mm pefabloc SC, 0.1 μg/ml leupeptin, 0.5 mm o-phenanthrolin, 0.5 μg/ml pepstatin A, 200 mm sorbitol, 10 mm PIPES/KOH, pH 6.8. This mixture was incubated for 1 h at 27 °C. For measuring luciferase uptake, the samples were chilled on ice, diluted with 300 μl of 150 mm KCl in PS buffer (200 mm sorbitol, 10 mm PIPES/KOH, pH 6.8), and centrifuged (6800 × g, 4 min, fixed angle table top centrifuge), and the pellet was washed once more with 150 mm KCl in PS buffer and resuspended in 50 μl 150 mm KCl in PS buffer. Proteinase K was added (0.3 mg/ml from 10× stock) and incubated on ice for 10 min. Digestion was stopped by adding 50 μl 1 mm phenylmethylsulfonyl fluoride, 150 mm KCl in PS buffer. Luciferase activity was determined using an assay kit according to the manufacturer's instructions (Berthold Detection Systems, Pforzheim, Germany); 25 μl of sample were mixed with 25 μl of lysis buffer and 25 μl of substrate A (containing ATP). 25 μl of substrate B (1 mm luciferin) were added directly before counting light emission in a microplate luminometer (LB 96 V, Berthold Technologies, Bad Wildbad, Germany). Alkaline phosphatase activity was determined in a 25-μl aliquot as described previously (6Sattler T. Mayer A. J. Cell Biol. 2000; 151: 529-538Crossref PubMed Scopus (88) Google Scholar). Uptake activity was calculated as the quotient of luciferase activity over alkaline phosphatase activity ((counts/s)/(ΔA405/min)) and referred to an uninhibited standard reaction (60 min, 27 °C), which was set to 100%. In some experiments, horseradish peroxidase (40 μg/ml) was used instead of luciferase. For activity determination, a 25-μl sample (vacuoles after washing and proteinase K digestion, in PS buffer plus 1% Triton X-100) was mixed in an opaque 96-well microtiter plate with 25 μl of substrate (5 mm luminol, 10 mm H2O2, 100 mm Tris/HCl, pH 8.5, freshly prepared) and 25 μl of cosubstrate (200 μm luciferin, 100 mm Tris/HCl, pH 8.5). Luminescence was measured in a microplate luminometer (LB 96 V, Berthold Technologies), and background activity (vacuoles without horseradish peroxidase) was subtracted. Uptake activity was calculated as described above. Thin Section Electron Microscopy—Yeast cells were cryofixed using a propane jet freezing device (JFD 030, Bal-Tec, Balzers AG) and freeze-substituted in 0.5% uranyl acetate in ethanol at –90 °C for 35 h, at –60 °C for 4 h, and –50 °C for 2 h in a freeze-substitution unit (FSU 010; Bal-Tec, Balzers AG). After washing with ethanol at –35 °C, the samples were infiltrated with Lowicryl HM20 and UV-polymerized at –35 °C for 48 h. Ultrathin sections stained with uranyl acetate and lead citrate were viewed in a Philips CM 10 electron microscope. One of the most important approaches to explore complex biochemical processes is kinetic analysis. A dissection into distinguishable reaction phases can serve to ascribe the interactions of relevant components and their dynamic changes to defined steps of the overall process and greatly facilitates the formulation of hypotheses on the molecular mechanism. Therefore, the identification of intermediates has always been an important and fruitful part in the analysis of complex biochemical reactions. In order to better understand vacuolar membrane invagination we sought to identify distinct reaction stages for this process. Since proteins and lipids involved in vacuolar membrane invagination are not yet known, we attempted to generate tools for kinetic analysis by identifying low molecular weight inhibitors. Such pharmacological approaches ("chemical genetics") have proven useful to dissect complex physiological phenomena (34Stockwell B.R. Nat. Rev. Gen. 2000; 1: 116-125Crossref PubMed Scopus (380) Google Scholar). If, as for vacuoles, sufficient quantities can be prepared, relevant target proteins for the inhibitors can even be identified by direct fractionation of the membrane on affinity matrices or by cross-linking approaches (35Peters C. Bayer M.J. Bühler S. Andersen J.S. Mann M. Mayer A. Nature. 2001; 409: 581-588Crossref PubMed Scopus (426) Google Scholar, 36Eitzen G. Thorngren N. Wickner W. EMBO J. 2001; 20: 5650-5656Crossref PubMed Scopus (95) Google Scholar, 37Müller O. Johnson D.I. Mayer A. EMBO J. 2001; 20: 5657-5665Crossref PubMed Scopus (65) Google Scholar, 38Kato M. Wickner W. EMBO J. 2001; 20: 4035-4040Crossref PubMed Scopus (125) Google Scholar, 39Peters C. Andrews P.D. Stark M.J.R. Cesaro-Tadic S. Glatz A. Podtelejnikov A. Mann M. Mayer A. Science. 1999; 285: 1084-1087Crossref PubMed Scopus (138) Google Scholar, 40Müller O. Bayer M. Peters C. Andersen S. Mann M. Mayer A. EMBO J. 2002; 21: 259-269Crossref PubMed Scopus (115) Google Scholar). In a pilot study, we screened 46 commercially available low molecular weight substances for inhibitory activity in the previously described (6Sattler T. Mayer A. J. Cell Biol. 2000; 151: 529-538Crossref PubMed Scopus (88) Google Scholar) in vitro microautophagy reaction. These compounds were selected because they target processes known to be involved in membrane transport, such as protein phosphorylation, lipid metabolism, membrane fluidity, and cytoskeleton rearrangements. The majority of the agents (26 of 46) did not influence microautophagy in vitro at all (Table I).Table ILow molecular weight inhibitors without effect on in vitro microautophagyInhibitorConcentration testedNocodazole200 μmColchicine1 mmInositol 1,4,5-trisphosphate500 μmAdenophostin50 μmR59949 (diacylglycerolkinase inhibitor)200 μmMethylarachidonoylfluorophosphonate500 μmFarnesyl transferase inhibitor (Alexis no. 290-005)2 mmPerillic acid1 mmOleic acid300 μmMyristoyllysophosphatidylcholine100 μmCholesterol2 mmCaffeine5 mm3-Methyladenine5 mmPhorbol 12-myristate 13-acetate1 mmKN62 (protein kinase inhibitor)1 mmKT5833 (protein kinase inhibitor)5000 μmH9 (protein kinase inhibitor)1 mmGDPβS1 mmNaF2 mmNa3VO4200 μmSodium pyrophosphate200 μmDeltamethrin1 mmCantharidine1 mmMicrocystin LR100 μmCalyculine A20 μmOcadaic acid20 μm Open table in a new tab 13 of the 46 agents showed effects on the in vitro uptake assay, but they were not pursued further, because they affected luciferase uptake only at unreasonably high concentrations, inhibited luciferase activity, or lysed vacuoles (Table II).Table IINonspecific inhibitors of in vitro microautophagyInhibitorSide effect and/or IC50EdelfosineIC50 = 100 μm; vacuole lysisCyclosporinIC50 = 100 μm; published IC50 = 10 nm (52Cardenas M.E. Lim E. Heitman J. J. Biol. Chem. 1995; 270: 20997-21002Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar)NeomycinIC50 = 250 μm; vacuoles difficult to resuspendU73122IC50 = 150 μm; maleimido group inactivates luciferaseManoalidIC50 = 5 μm; vacuoles cannot be sedimentedPodophyllotoxinIC50 = 250 μm; published IC50 = 12 nm (53Gupta R.S. Cancer Res. 1983; 43: 505-512PubMed Google Scholar)PaclitaxelIC90>500 μm; published IC50 = 12 nm (53Gupta R.S. Cancer Res. 1983; 43: 505-512PubMed Google Scholar)Polymyxin BIC90 = 500 μm; detergent-like properties at this concentrationStaurosporinIC50 = 200 μm; published IC50 = 2.7 nm (54Tamaoki T. Biochim. Biophys. Res. Commun. 1986; 135: 397-402Crossref PubMed Scopus (2224) Google Scholar)MellitinVacuole lysis above 10 μmF48/80Inhibition also if added after uptake reactionPhospholipase A2Vacuole lysisPhenylarsinoxideInhibits luciferase by inactivating SH-groups Open table in a new tab Seven of the substances (Table III) showed nearly complete inhibition of luciferase uptake without affecting vacuolar integrity, as judged by light microscopy and by reversibility of inhibitor action (see below) (see Fig. 4). None of these inhibitors had any effect if added after the uptake reaction, confirming that vacuoles were not lysed by the inhibitors.Table IIIInhibitors of in vitro microautophagyInhibitors and their effect in other model systemsEffect on in vitro microautophagyIC50IC90GTPγS; inhibits in vitro vacuole fusion at 1 mm (35Peters C. Bayer M.J. Bühler S. Andersen J.S. Mann M. Mayer A. Nature. 2001; 409: 581-588Crossref PubMed Scopus (426) Google Scholar)200 μm1 mmFCCP; dissipates vacuolar membrane potential at 10 μm (35Peters C. Bayer M.J. Bühler S. Andersen J.S. Mann M. Mayer A. Nature. 2001; 409: 581-588Crossref PubMed Scopus (426) Google Scholar)2 μm10 μmNystatin; inhibits S. cerevisiae growth at 7 μm (55Fryberg M. Oehlschlager A.C. Unrau A.M. Arch. Biochem. Biophys. 1974; 160: 83-89Crossref PubMed Scopus (50) Google Scholar)1 μm3 μmAmphotericin B; same action and potency as nystatin (47Bhuiyan M.S.A. Ito Y. Nakamura A. Tanaka N. Fujita K. Fukui H. Takegawa K. Biosci. Biotechnol. Biochem. 1999; 63: 1075-1082Crossref PubMed Scopus (15) Google Scholar)2 μm5 μmRapamycin; induces macroautophagy at 0.2 μm (56Noda T. Ohsumi Y. J. Biol. Chem. 1998; 273: 3963-3966Abstract Full Text Full Text PDF PubMed Scopus (1052) Google Scholar)20 μm40 μmK252a; inhibits hepatic autophagy with IC50 = 30 μm (57Holen I. Gordon P.B. Seglen P.O. Eur. J. Biochem. 1993; 215: 113-122Crossref PubMed Scopus (77) Google Scholar)50 μm200 μmAristolochic acid; inhibits phospholipase A2, IC50 = 400 μm (58Rosenthal M.D. Vishwanath B.S. Franson R.C. Biochim. Biophys. Acta. 1989; 1001: 1-8Crossref PubMed Scopus (88) Google Scholar)250 μm750 μm Open table in a new tab Inhibition of luciferase uptake by these drugs depended on their concentration (Fig. 1a) but also on the presence of cytosol. Type I microautophagy in vitro is strongly stimulated by the addition of cytosol (6Sattler T. Mayer A. J. Cell Biol. 2000; 151: 529-538Crossref PubMed Scopus (88) Google Scholar). In reactions lacking cytosol, uptake persisted, albeit only with 20% of the activity of the control samples. Under these conditions, none of the agents listed in Table I impaired uptake any more (Fig. 1b). Uptake in the absence of cytosol was still ATP-dependent, indicating that the signal measured had been generated by an energy-dependent process. Since the action of all inhibitors tested required cytosol, these substances might prevent binding of cytosolic factors to the vacuolar membrane. The absence of effects of microtubule-directed drugs such as nocodazole and colchicine is notable, since vacuoles are very large organelles and occupy up to a third of the total cell volume. Our favored hypothesis on vacuolar membrane invagination had been that long filaments, such as microtubules, might push toward the vacuolar lumen, deforming the vacuolar boundary membrane. This hypothesis appeared particularly attractive, since microtubules are very dynamic structures in yeast, growing and shrinking vigorously in the course of the cell cycle. Furthermore, they can be close to the vacuolar membrane (41Carminati J.L. Stearns T. J. Cell Biol. 1997; 138: 629-641Crossref PubMed Scopus (402) Google Scholar). Microtubule-destabilizing agents did not inhibit vacuole invagination in vitro (Fig. 2a), even at concentrations high enough to suppress growth of intact cells by disassembly of the spindle (42Kirisako T. Baba M. Ishihara N. Miyazawa K. Ohsumi M. Yoshinori T. Noda T. Ohsumi Y. J. Cell Biol. 1999; 147: 435-446Crossref PubMed Scopus (719) Google Scholar). 2A. Mayer, unpublished observation. This suggested that vacuolar membrane invagination can occur independently of microtubules. Electron microscopic analysis of starved yeast cells (Fig. 2b) supported this conclusion. Cytoplasmic microtubules in yeast are organized exclusively by the spindle pole body. They emanate directly from this structure and are generally very short (43Botstein D. Amberg D. Mulholland J. Huffaker T. Adams A. Drubin D. Stearns T. Pringle J.R. Broach J.R. Jones E.W. The Molecular and Cellular Biology of the Yeast Saccharomyces cerevisiae. Vol. 3. Cold Spring Harbor Laboratory Press, Plainview, NY1997: 1-90Google Scholar). If cytoplasmic microtubules initiated vacuolar invaginations, the spindle pole bodies should be close to the site of invagination. However, microautophagic membrane invaginations could be observed on parts of the vacuolar membrane not facing the nucleus and the spindle pole body. Hence, vacuolar invaginations can obviously develop without the aid of microtubules. Using the pharmacological inhibitors identified (Table I), we could now attempt a kinetic analysis of type I microautophagy. In order to test whether the inhibitors affected different stages of vacuolar invagination, a standard uptake reaction was started. Inhibitors were added at different time points (Fig. 3), and the reaction was continued until the end of a standard incubation period. Agents influencing very early steps of type I microautophagy would be ineffective if added late during the reaction, whereas agents acting on late events of microautophagy would be active throughout the reaction. As a control, samples were transferred to ice, a treatment that blocks many processes depending on membrane or protein dynamics (44Balch W.E. Rothman J.E. Arch. Biochem. Biophys. 1985; 240: 413-425Crossref PubMed Scopus (210) Google Scholar, 45Baker D. Schekman R. Methods Cell Biol. 1989; 31: 127-141Crossref PubMed Scopus (16) Google Scholar). Inhibitors could be grouped into two categories according to their kinetic properties. Inhibition curves of FCCP/valinomycin, rapamycin, and K252a developed almost in parallel to each other, crossing the ice curve at 35 min and keeping their inhibitory potential throughout the incubation (Fig. 3, a, c, and e). We designate these as late acting (class B) inhibitors. In contrast, the reaction had become largely resistant to class A inhibitors (GTPγS, nystatin, and aristolochic acid) after 30 min, resulting in a curve convex toward the top (Fig. 3, b, d, and f). The ice curve showed a very shallow slope during the first 30 min but a steep rise between 30 and 45 min, indicating that most of the luciferase uptake had taken place during this relatively short period of time. Inhibition by transfer on ice became ineffective late during the reaction because, after 45 min, class B inhibitors (FCCP/valinomycin, rapamycin, and K252a) still blocked further luciferase uptake efficiently, but transfer on ice did not. The kinetics shown thus define three reaction states: 1) GTPγS-, nystatin-, and aristolochic acid-sensitive; 2) cold-sensitive; 3) FCCP/valinomycin-, rapamycin-, and K252a-sensitive. In order to exclude the possibility that the inhibitors had damaged the vacuoles nonspecifically, we tested whether an inhibitory block could be reversed by removal of the inhibitor. Two-step reactions were performed. During a first 30-min incubation, inhibitors were present in a complete microautophagy reaction. The agents were then removed by sedimenting the vacuoles, discarding the supernatant, and resuspending the membranes in fresh medium. Aliquots of these samples were either set on ice or incubated at 27 °C in the presence or absence of an inhibitor. This second incubation was stopped after 60 min, and luciferase uptake was determined in all samples (Fig. 4). During the first incubation without inhibitor at 27 °C, only about 20% of the total signal was generated, consistent with the very shallow slope of the ice curve during this period of time (Fig. 3). All inhibitors were reversible by reisolating and washing the membranes, except nystatin (Fig. 4g). The yellow nystatin partitions into membranes (46Bolard J. Biochim. Biophys. Acta. 1986; 864: 257-304Crossref PubMed Scopus (690) Google Scholar) and is retained by vacuoles even after reisolation, as evident from the intense staining of the organelles after nystatin treatment (not shown). Uptake was almost completely abolished if the same inhibitors as in the first incubation were added to the second stage (bar 2). This indicates that the membranes had not passed the initial block during reisolation and resuspension. Vacuoles lost about half of their uptake potential if the first incubation was performed either on ice or in the presence of inhibitor (Fig. 4, compare panel b and panels a and c–h) (i.e. under all conditions that prevented uptake). The reason for this effect is unclear. It is possible that the addition of ATP and cytosol renders accessory vacuolar components more labile and prone to inactivation if the reaction cannot proceed. Next, we tested whether there would be stable reaction intermediates. If such intermediates existed, the reaction step leading to these intermediates should be essentially irreversible. Then it should be possible to accumulate these intermediates in the presence of late acting (class B) in
Referência(s)