Cargo Proteins Facilitate the Formation of Transport Vesicles in the Cytoplasm to Vacuole Targeting Pathway
2004; Elsevier BV; Volume: 279; Issue: 29 Linguagem: Inglês
10.1074/jbc.m404399200
ISSN1083-351X
AutoresTakahiro Shintani, Daniel J. Klionsky,
Tópico(s)Studies on Chitinases and Chitosanases
ResumoSelective incorporation of cargo proteins into the forming vesicle is an important aspect of protein targeting via vesicular trafficking. Based on the current paradigm of cargo selection in vesicular transport, proteins to be sorted to other organelles are condensed at the vesicle budding site in the donor organelle, a process that is mediated by the interaction between cargo and coat proteins, which constitute part of the vesicle forming machinery. The cytoplasm to vacuole targeting (Cvt) pathway is an unconventional vesicular trafficking pathway in yeast, which is topologically and mechanistically related to autophagy. Aminopeptidase I (Ape1) is the major cargo protein of the Cvt pathway. Unlike the situation in conventional vesicular transport, precursor Ape1, along with its receptor Atg19/Cvt19, is packed into a huge complex, termed a Cvt complex, independent of the vesicle formation machinery. The Cvt complex is subsequently incorporated into the forming Cvt vesicle. The deletion of APE1 or ATG19 compromised the organization of the pre-autophagosomal structure (PAS), a site that is thought to play a critical role in Cvt vesicle/autophagosome formation. The proper organization of the PAS also required Atg11/Cvt9, a protein that localizes the cargo complex at the PAS. Accordingly, the deletion of APE1, ATG19, or ATG11 affected the formation of Cvt vesicles. These observations suggest a unique concept; in the case of the Cvt pathway, the cargo proteins facilitate receptor recruitment and vesicle formation rather than the situation with most vesicular transport, in which the forming vesicle concentrates the cargo proteins. Selective incorporation of cargo proteins into the forming vesicle is an important aspect of protein targeting via vesicular trafficking. Based on the current paradigm of cargo selection in vesicular transport, proteins to be sorted to other organelles are condensed at the vesicle budding site in the donor organelle, a process that is mediated by the interaction between cargo and coat proteins, which constitute part of the vesicle forming machinery. The cytoplasm to vacuole targeting (Cvt) pathway is an unconventional vesicular trafficking pathway in yeast, which is topologically and mechanistically related to autophagy. Aminopeptidase I (Ape1) is the major cargo protein of the Cvt pathway. Unlike the situation in conventional vesicular transport, precursor Ape1, along with its receptor Atg19/Cvt19, is packed into a huge complex, termed a Cvt complex, independent of the vesicle formation machinery. The Cvt complex is subsequently incorporated into the forming Cvt vesicle. The deletion of APE1 or ATG19 compromised the organization of the pre-autophagosomal structure (PAS), a site that is thought to play a critical role in Cvt vesicle/autophagosome formation. The proper organization of the PAS also required Atg11/Cvt9, a protein that localizes the cargo complex at the PAS. Accordingly, the deletion of APE1, ATG19, or ATG11 affected the formation of Cvt vesicles. These observations suggest a unique concept; in the case of the Cvt pathway, the cargo proteins facilitate receptor recruitment and vesicle formation rather than the situation with most vesicular transport, in which the forming vesicle concentrates the cargo proteins. The localization of many intracellular proteins is dependent upon movement within transient transport vesicles. The precise transport of proteins via vesicular trafficking is guaranteed by the selective incorporation of cargo into the forming vesicles and specific membrane fusion with an acceptor organelle. The cytoplasm to vacuole targeting (Cvt) 1The abbreviations used are: Cvt, cytoplasm to vacuole targeting; PAS, pre-autophagosomal structure; GFP, green fluorescent protein; PI, phosphatidylinositol; YFP, yellow fluorescent protein; PE, phosphatidylethanolamine; Ape1, aminopeptidase I; Ams1, α-mannosidase; prApe1, precursor Ape1. pathway is an autophagy-related protein targeting pathway in yeast, whereby the resident vacuolar hydrolases, aminopeptidase I (Ape1) and α-mannosidase (Ams1), are directly targeted from the cytoplasm to the vacuole. In this pathway, these two hydrolases are selectively enwrapped by double membrane-bound vesicles, termed Cvt vesicles, followed by fusion with the vacuolar membrane and a breakdown of the inner membrane structure to release precursor Ape1 (prApe1) and Ams1 into the vacuolar lumen (1Klionsky D.J. Cueva R. Yaver D.S. J. Cell Biol. 1992; 119: 287-299Crossref PubMed Scopus (307) Google Scholar, 2Hutchins M.U. Klionsky D.J. J. Biol. Chem. 2001; 276: 20491-20498Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). The process of the Cvt pathway and autophagy are topologically and mechanistically similar, even though they are different in their physiological functions (3Takeshige K. Baba M. Tsuboi S. Noda T. Ohsumi Y. J. Cell Biol. 1992; 119: 301-311Crossref PubMed Scopus (969) Google Scholar, 4Baba M. Osumi M. Scott S.V. Klionsky D.J. Ohsumi Y. J. Cell Biol. 1997; 139: 1687-1695Crossref PubMed Scopus (280) Google Scholar, 5Scott S.V. Baba M. Ohsumi Y. Klionsky D.J. J. Cell Biol. 1997; 138: 37-44Crossref PubMed Scopus (142) Google Scholar). Autophagy is a cellular process responsible for the non-selective bulk degradation of cytoplasmic components in eukaryotic cells, which is induced in response to environmental cues such as nutrient starvation and hormonal stimuli (6Klionsky D.J. Ohsumi Y. Annu. Rev. Cell Dev. Biol. 1999; 15: 1-32Crossref PubMed Scopus (391) Google Scholar, 7Klionsky D.J. Emr S.D. Science. 2000; 290: 1717-1721Crossref PubMed Scopus (3014) Google Scholar). In yeast, nutrient starvation induces the formation of autophagosomes, which are much larger (300–900 nm in diameter) than Cvt vesicles (150 nm in diameter), for efficient transport of cytoplasm to the vacuole. On the other hand, the Cvt pathway is a constitutive biosynthetic pathway highly specific for prApe1 and Ams1. The cargo specificity in the Cvt pathway is conferred by Atg19/Cvt19 that has been characterized as a cargo receptor (8Scott S.V. Guan J. Hutchins M.U. Kim J. Klionsky D.J. Mol. Cell. 2001; 7: 1131-1141Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). Recently, we elucidated the mechanism of cargo selection in the Cvt pathway (9Shintani T. Huang W.-P. Stromhaug P.E. Klionsky D.J. Dev. Cell. 2002; 3: 825-837Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar). Two independent processes contributed to the selective incorporation of prApe1 into the Cvt vesicle: the self-assembly of the prApe1 complex, and its recruitment to the perivacuolar vesicle-forming site called the pre-autophagosomal structure (PAS). After synthesis of the prApe1 polypeptide, it rapidly forms dodecamers in the cytosol (10Kim J. Scott S.V. Oda M.N. Klionsky D.J. J. Cell Biol. 1997; 137: 609-618Crossref PubMed Scopus (117) Google Scholar), which further assemble into a higher order structure termed the Ape1 complex; assembly of the Ape1 complex is dependent on the prApe1 propeptide. The Ape1 complex recruits Atg19 through the interaction between the propeptide of prApe1 and a coiled-coil motif of Atg19. Atg11/Cvt9 then binds to the C terminus of Atg19 to target the cargo-receptor complex to the PAS, where the interactions between Atg19 and PAS components ensure the incorporation of the cargo complex into the Cvt vesicle (9Shintani T. Huang W.-P. Stromhaug P.E. Klionsky D.J. Dev. Cell. 2002; 3: 825-837Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar). Unlike most receptors that cycle between donor and acceptor membranes, Atg19 is targeted to the vacuole together with cargo proteins and degraded there. The simultaneous binding of another cargo molecule, Ams1, to Atg19 results in an accumulation of Ams1 on the Ape1 complex, allowing an efficient transport of Ams1 to the vacuole. Interestingly, the lack of an Ape1 complex resulted in a dramatic decrease in the turnover of Atg19 as well as a decrease of Ams1 transport to the vacuole (8Scott S.V. Guan J. Hutchins M.U. Kim J. Klionsky D.J. Mol. Cell. 2001; 7: 1131-1141Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, 9Shintani T. Huang W.-P. Stromhaug P.E. Klionsky D.J. Dev. Cell. 2002; 3: 825-837Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar), suggesting that the Ape1 complex facilitates the incorporation of an Atg19-Ams1 complex into the Cvt vesicles. Because the turnover of Atg19 is dependent on Cvt vesicle formation (8Scott S.V. Guan J. Hutchins M.U. Kim J. Klionsky D.J. Mol. Cell. 2001; 7: 1131-1141Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar), these results raised a question as to whether the Cvt cargo could also induce Cvt vesicle formation or whether vesicle formation was constitutive and empty vesicles could be formed even without cargo proteins. The role of cargo proteins in facilitating or directing vesicle formation has been difficult to study because in most vesicular trafficking events the full range of cargo molecules have not been defined; in the Cvt pathway, prApe1 appears to be the predominant cargo protein, and even Ams1 is a relatively minor constituent. Even though the Ape1 complex is selectively incorporated into autophagosomes under starvation conditions, neither Ape1 nor Atg19 is required for autophagy (8Scott S.V. Guan J. Hutchins M.U. Kim J. Klionsky D.J. Mol. Cell. 2001; 7: 1131-1141Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, 11Suzuki K. Kamada Y. Ohsumi Y. Dev. Cell. 2002; 3: 815-824Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar), suggesting that autophagosome formation does not depend on the presence of specific cargo components. In this study, we show that the cargo-receptor complex facilitates the formation of Cvt vesicles but not autophagosomes. Strains, Plasmids, and Media—The Saccharomyces cerevisiae yeast strains used in this study are listed in Table I. A plasmid expressing GFP-Atg8 was based on pRS306 containing the GFP-ATG8 gene with the endogenous ATG8 promoter (12Suzuki K. Kirisako T. Kamada Y. Mizushima N. Noda T. Ohsumi Y. EMBO J. 2001; 20: 5971-5981Crossref PubMed Scopus (806) Google Scholar) and used for yeast transformation to integrate GFP-ATG8 at the URA3 locus. The pRS316 GFP-APG1 plasmid and YCplac33 GFP-APG2 (pTS112) (13Shintani 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) were kind gifts from Dr. Yoshinori Ohsumi (National Institute for Basic Biology, Okazaki, Japan). The atg1ts allele (12Suzuki K. Kirisako T. Kamada Y. Mizushima N. Noda T. Ohsumi Y. EMBO J. 2001; 20: 5971-5981Crossref PubMed Scopus (806) Google Scholar) was cloned on pRS414. Yeast cells were grown in SCD medium (0.67% yeast nitrogen base without amino acids, 0.5% casamino acid, and 2% glucose) supplemented with 0.003% adenine, 0.005% tryptophan, and 0.002% uracil, if necessary. For nitrogen starvation, SD(–N) medium (0.17% yeast nitrogen base without ammonium sulfate and amino acids and 2% glucose) was used.Table IYeast strains used in this studyStrainGenotypeSourceSEY6210MATα his3-Δ200 leu2-3,112 lys2-801 trp1-Δ901 ura3-52 suc2-Δ9 GALRef. 34Robinson J.S. Klionsky D.J. Banta L.M. Emr S.D. Mol. Cell. Biol. 1988; 8: 4936-4948Crossref PubMed Scopus (739) Google ScholarFRY138SEY6210; ATG9-YFP::HIS5 S.p. atg1Δ::URA3Ref. 27Reggiori F. Tucker K.A. Stromhaug P.E. Klionsky D.J. Dev. Cell. 2004; 6: 79-90Abstract Full Text Full Text PDF PubMed Scopus (377) Google ScholarTYY014SEY6210; ATG20-YFP::HIS5 S.p. vps38Δ::LEU2 K.l. atg11Δ::URA3 K.l.This studyYTS135SEY6210; ATG9-YFP::HIS5 S.p. atg1Δ::URA3 atg19Δ::LEU2 K.l.This studyYTS136SEY6210; ATG9-YFP::HIS5 S.p. atg1Δ::URA3 ape1Δ::LEU2This studyYTS150SEY6210; ATG9-YFP::HIS5 S.p. atg1Δ::URA3 atg11Δ::LEU2 K.l.This studyYTS180SEY6210; ATG20-YFP::HIS5 S.p. vps38Δ::LEU2 K.l.This studyYTS184SEY6210; ATG20-YFP::HIS5 S.p. vps38Δ::LEU2 K.l. atg19Δ::URA3 K.l.This studyYTS185SEY6210; ATG20-YFP::HIS5 S.p. vps38Δ::LEU2 K.l. ape1Δ::TRP1This studyYTS187SEY6210; URA3::GTP-ATG8This studyYTS188SEY6210; atg1Δ::HIS5 S.p. GFP-ATG8:: URA3This studyYTS189SEY6210; pep4Δ:: LEU2 GFP-ATG8::URA3This studyYTS190SEY6210; atg19Δ::HIS5 S.p. GFP-ATG8::URA3This studyYTS191SEY6210; ape1Δ::LEU2 GFP-ATG8::URA3This studyYTS192SEY6210; atg11Δ::HIS3 GFP-ATG8::URA3This studyYTS193SEY6210; atg2Δ::HIS5 S.p. [GFP-ATG2 URA3]This studyYTS194SEY6210; atg2Δ::HIS5 S.p. atg19Δ::LEU2 K.l. [GFP-ATG2 URA3]This studyYTS195SEY6210; atg2Δ::HIS5 S.p. ape1Δ::LEU2 [GFP-ATG2 URA3]This studyYTS196SEY6210; atg2Δ::HIS5 S.p. atg11Δ::LEU2 K.l. [GFP-ATG2 URA3]This studyYTS197SEY6210 [GFP-ATG1 URA3]This studyYTS198SEY6210; atg19Δ::HIS5 S.p. [GFP-ATG1 URA3]This studyYTS199SEY6210; ape1Δ::LEU2 [GFP-ATG1 URA3]This studyYTS200SEY6210; atg11Δ::HIS3 [GFP-ATG1 URA3]This studyYTS201SEY6210; atg1Δ::HIS5 S.p. URA3::GFP-ATG8 [atg1ts TRP1]This studyYTS202SEY6210; atg1Δ::HIS5 S.p. atg19Δ::LEU2 K.l. URA3::GFP-ATG8 [atg1ts TRP1]This studyYTS203SEY6210; atg1Δ::HIS5 S.p. ape1Δ::LEU2 URA3::GFP-ATG8 [atg1ts TRP1]This studyYTS204SEY6210; atg1Δ::HIS5 S.p. atg11Δ::LEU2 K.l. URA3::GFP-ATG8 [atg1ts TRP1]This study Open table in a new tab Transport of GFP-Atg8 to the Vacuole—The strains harboring the atg1ts allele were grown in SCD medium at 37 °C overnight to A600 = 1.0. The cultures were divided into two tubes, and either rapamycin (0.2 μg/ml) or the drug vehicle was added to each tube. After incubation at 37 °C for 10 min, the tubes were placed at 30 °C to activate the Cvt pathway or autophagy. At various time points, 1 ml of culture was harvested and used to prepare a protein extract. Protein extracts equivalent to A600 = 0.1 unit of yeast cells were subjected to SDS-PAGE and probed with anti-Ape1 antiserum and anti-GFP antibody (Covance Research Products, Berkeley, CA). Fluorescence Microscopy—Yeast cells expressing fluorescent proteinfused chimeras were grown in SCD medium to mid-log phase. For nitrogen starvation, the cells grown in SCD medium were washed with water twice and resuspended in SD(–N) medium. The cells were observed with a fluorescence microscope as described previously (14Kim J. Huang W.-P. Stromhaug P.E. Klionsky D.J. J. Biol. Chem. 2002; 277: 763-773Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). Atg8 Can Be Used to Trace Cvt Vesicle Formation—Atg19 is a receptor protein for the vacuolar hydrolases Ape1 and Ams1 in the Cvt pathway. It is transported to the vacuole together with cargo proteins and degraded within the vacuole lumen. Previously, we found that the turnover of Atg19 and the transport of Ams1 were severely affected by a lack of Ape1 (9Shintani T. Huang W.-P. Stromhaug P.E. Klionsky D.J. Dev. Cell. 2002; 3: 825-837Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar). Because both Atg19 turnover and Ams1 transport are also dependent on the proper function of the Cvt pathway, we asked whether the Ape1 complex could induce the formation of Cvt vesicles or whether empty vesicles could be formed without the Ape1 complex. To analyze the effect of a lack of cargo complex on the formation of Cvt vesicles, we needed to develop a suitable marker protein. Precursor Ape1 is the standard marker used to monitor delivery via the Cvt and autophagy pathways; however, we were unable to use Ape1 as a marker because our goal was to examine vesicle formation in the absence of this specific cargo protein. Among the characterized Atg proteins, only Atg19 and Atg8/Aut7 remain associated with the completed Cvt vesicle/autophagosome. Atg19 was not suitable as a marker protein because it functions in cargo recognition. Accordingly, we used Atg8 as a marker of the Cvt pathway. Atg8 is essential for Cvt vesicle formation and for expansion of the autophagosome membrane (15Kirisako T. Baba M. Ishihara N. Miyazawa K. Ohsumi M. Yoshimori T. Noda T. Ohsumi Y. J. Cell Biol. 1999; 147: 435-446Crossref PubMed Scopus (719) Google Scholar, 16Huang W.-P. Scott S.V. Kim J. Klionsky D.J. J. Biol. Chem. 2000; 275: 5845-5851Abstract Full Text Full Text PDF PubMed Scopus (199) 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). Similar to Atg19, Atg8 is delivered to the vacuole as a component of the Cvt vesicle/autophagosome and is degraded after breakdown of the inner vesicle within the vacuole lumen. Accordingly, Cvt vesicle/autophagosome formation can be assessed by measuring the amount of Atg8 protein delivered to the vacuole. Using Atg8 to monitor vesicle formation, however, presented a significant problem for analyzing the Cvt pathway; Atg8 is synthesized at very low levels under vegetative conditions and is only induced upon starvation. Accordingly, it is problematic to monitor delivery of Atg8 under vegetative conditions through a radioactive pulse/chase analysis; the level of protein delivered to the vacuole is too low to allow an accurate quantification, and it is inherently difficult to follow the kinetics of vacuolar delivery by examining loss of a protein. Because of these technical difficulties, we designed an alternate strategy to monitor vesicle formation. GFP tagging of Atg8 has been used to trace the process of the Cvt pathway and autophagy (12Suzuki K. Kirisako T. Kamada Y. Mizushima N. Noda T. Ohsumi Y. EMBO J. 2001; 20: 5971-5981Crossref PubMed Scopus (806) Google Scholar, 18Kim J. Huang W.-P. Klionsky D.J. J. Cell Biol. 2001; 152: 51-64Crossref PubMed Scopus (188) Google Scholar). Even though Atg8 is subject to degradation upon vacuolar delivery, the relative stability of GFP to the activity of vacuolar hydrolases led us to propose that the GFP moiety would accumulate in the vacuolar lumen as a consequence of the transport of GFP-Atg8 to the vacuole; the accumulated GFP could be easily monitored and would reflect delivery of Atg8. To confirm the suitability of this approach, we performed immunoblot analysis of GFP-Atg8 using anti-GFP antibody. In wild type cells expressing GFP-Atg8 grown in rich medium, we detected protein bands of 40 and 26 kDa (Fig. 1A, bottom panel). These molecular masses correspond to the predicted sizes of full-length GFP-Atg8 and free GFP. As controls, we examined atg1Δ/agp1Δ and pep4Δ strains, which are defective in Cvt vesicle/autophagosome formation and the breakdown of intravacuolar vesicles, respectively. In contrast to the result seen in wild type cells, only full-length GFP-Atg8 was seen in the mutant strains (Fig. 1A, bottom panel). These results indicated that the generation of free GFP was dependent on the proper function of the Cvt pathway. When autophagy was induced by incubating the cells in starvation medium, the transport of GFP-Atg8 was enhanced in wild type cells, whereas again no liberation of GFP was observed in atg1Δ or pep4Δ mutant cells (Fig. 1A, bottom panel). These results support the idea that an increased level of Atg8 is required to form autophagosomes (17Abeliovich H. Dunn Jr., W.A. Kim J. Klionsky D.J. J. Cell Biol. 2000; 151: 1025-1034Crossref PubMed Scopus (236) Google Scholar). An analysis of Ape1 from the same protein extracts verified that the mutant strains displayed the expected phenotypes with regard to prApe1 processing (Fig. 1A, top panel). To allow a kinetic analysis of GFP-Atg8 transport, we took advantage of a conditional atg1 mutant. Cells harboring a temperature-sensitive allele of ATG1, atg1ts, and expressing GFP-Atg8 were grown to mid-log phase at non-permissive temperature and then shifted to permissive temperature to activate the Cvt pathway, followed by immunoblot analysis with anti-Ape1 antiserum and anti-GFP antibody. After shifting to the permissive temperature, prApe1 was converted to the mature form (mApe1) within 1 h, indicating that prApe1 reached the vacuole immediately after the atg1ts block was released (Fig. 1B, top left, top panel). Similarly, the conversion of GFP-Atg8 to GFP occurred within 1 h after incubation at 30 °C, and accumulation of GFP gradually increased during the time course (Fig. 1B, top left, bottom panel). The addition of rapamycin to the culture, which mimics nutrient starvation conditions, resulted in an enhanced transport of GFP-Atg8 to the vacuole (Fig. 1B, top left, bottom panel). These results confirmed that this system is useful for a kinetic analysis of Cvt vesicle/autophagosome formation. Absence of the Cvt Cargo-Receptor Complex Impairs Cvt Vesicle but Not Autophagosome Formation—To investigate whether the Cvt cargo complex is involved in Cvt vesicle formation, we carried out kinetic analyses using an atg1ts ape1Δ double mutant strain expressing GFP-Atg8. After growth at 37 °C, the cells were shifted to 30 °C to activate the Cvt pathway. In the absence of rapamycin treatment, the generation of free GFP was severely delayed up to 4 h after incubation at 30 °C, suggesting that prApe1 is required for proper or efficient Cvt vesicle formation (Fig. 1B, bottom left, bottom panel). The receptor protein Atg19 is concentrated on the Ape1 complex and behaves like an adaptor protein to connect the Ape1 complex and the vesicle forming machinery; the Atg19-Ape1 complex is termed the Cvt complex. Thus, we hypothesized that Atg19 might be also important for Cvt vesicle formation. To test this, we carried out the same analysis with an atg1ts atg19Δ strain. As expected, GFP-Atg8 transport was delayed in this strain and showed kinetics similar to that seen in the atg1ts ape1Δ strain (Fig. 1B, top right, bottom panel). These results suggested that the cargo-receptor complex induces the formation of the Cvt vesicle rather than being incorporated within a constitutively forming vesicle. In contrast to the block in Cvt vesicle formation seen in rich medium, the deletion of either APE1 or ATG19 did not affect autophagosome formation when cells were treated with rapamycin to induce autophagy (Fig. 1B, bottom left and top right, bottom panels). This result was consistent with previous observations obtained by using autophagy-dependent activation of alkaline phosphatase activity (8Scott S.V. Guan J. Hutchins M.U. Kim J. Klionsky D.J. Mol. Cell. 2001; 7: 1131-1141Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, 11Suzuki K. Kamada Y. Ohsumi Y. Dev. Cell. 2002; 3: 815-824Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). It is believed that Cvt vesicles and autophagosomes are formed at the PAS, where most of the Atg proteins localize (11Suzuki K. Kamada Y. Ohsumi Y. Dev. Cell. 2002; 3: 815-824Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 12Suzuki K. Kirisako T. Kamada Y. Mizushima N. Noda T. Ohsumi Y. EMBO J. 2001; 20: 5971-5981Crossref PubMed Scopus (806) Google Scholar, 19Noda T. Suzuki K. Ohsumi Y. Trends Cell Biol. 2002; 12: 231-235Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). The Cvt complex also localizes to this structure (9Shintani T. Huang W.-P. Stromhaug P.E. Klionsky D.J. Dev. Cell. 2002; 3: 825-837Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, 11Suzuki K. Kamada Y. Ohsumi Y. Dev. Cell. 2002; 3: 815-824Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). To examine whether proper localization of the cargo-receptor complex to the PAS is also important for vesicle formation, we constructed an atg1ts atg11Δ/cvt9Δ strain expressing GFP-Atg8. Atg11/Cvt9 is a specific factor for the Cvt pathway and pexophagy (20Kim J. Kamada Y. Stromhaug P.E. Guan J. Hefner-Gravink A. Baba M. Scott S.V. Ohsumi Y. Dunn Jr., W.A. Klionsky D.J. J. Cell Biol. 2001; 153: 381-396Crossref PubMed Scopus (218) Google Scholar) and is required to localize the cargo-receptor complex to the PAS in the Cvt pathway (9Shintani T. Huang W.-P. Stromhaug P.E. Klionsky D.J. Dev. Cell. 2002; 3: 825-837Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar). In this strain, no transport of GFP-Atg8 was observed without rapamycin treatment, whereas with rapamycin the transport of GFP-Atg8 was comparable with that of other strains including the wild type (Fig. 1B). This result suggested that cargo recruitment to the PAS was also important for Cvt vesicle formation but not for autophagosome formation. Examination of Ape1 again confirmed the expected phenotype of the various strains. In particular, prApe1 was absent in the ape1Δ strain, showed normal maturation in the atg1ts strain at the permissive temperature, accumulated in the absence of Atg19, and was partially matured in the atg11Δ strain only under starvation conditions (Fig. 1B, top panels). The Cvt Complex Is Important for PAS Organization under Growing Conditions—The PAS is thought to be an organizing center for Cvt vesicle/autophagosome formation based on several lines of evidence: (i) most of the Atg proteins are located at the PAS under both growing and starvation conditions (12Suzuki K. Kirisako T. Kamada Y. Mizushima N. Noda T. Ohsumi Y. EMBO J. 2001; 20: 5971-5981Crossref PubMed Scopus (806) Google Scholar, 14Kim J. Huang W.-P. Stromhaug P.E. Klionsky D.J. J. Biol. Chem. 2002; 277: 763-773Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 19Noda T. Suzuki K. Ohsumi Y. Trends Cell Biol. 2002; 12: 231-235Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar); (ii) the localization of some Atg proteins at the PAS is dependent on the function of other Atg proteins (12Suzuki K. Kirisako T. Kamada Y. Mizushima N. Noda T. Ohsumi Y. EMBO J. 2001; 20: 5971-5981Crossref PubMed Scopus (806) Google Scholar, 13Shintani 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, 18Kim J. Huang W.-P. Klionsky D.J. J. Cell Biol. 2001; 152: 51-64Crossref PubMed Scopus (188) Google Scholar, 21Guan J. Stromhaug P.E. George M.D. Habibzadegah-Tari P. Bevan A. Dunn Jr., W.A. Klionsky D.J. Mol. Biol. Cell. 2001; 12: 3821-3838Crossref PubMed Scopus (154) Google Scholar); and (iii) GFP-Atg8 transiently localizes at the PAS and is then transported to the vacuole (12Suzuki K. Kirisako T. Kamada Y. Mizushima N. Noda T. Ohsumi Y. EMBO J. 2001; 20: 5971-5981Crossref PubMed Scopus (806) Google Scholar, 14Kim J. Huang W.-P. Stromhaug P.E. Klionsky D.J. J. Biol. Chem. 2002; 277: 763-773Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). Accordingly, we decided to examine the effect of depletion of the cargo complex on the organization of the PAS. First, we examined the localization of GFP-Atg8 under growing conditions. In a wild type strain, 25% of the cells contained a single GFP-Atg8 punctate signal or a few GFP-Atg8 punctate signals at the PAS (Fig. 2). In contrast, the PAS localization of GFP-Atg8 was observed in only 6.0% and 2.5% of ape1Δ and atg19Δ cells, respectively (Fig. 2). The atg11Δ strain also showed a severe defect in GFP-Atg8 localization at the PAS; only 3.3% of the cells had punctate signals of GFP-Atg8 (Fig. 2). These results suggested that the cargo-receptor complex and its recruitment to the PAS were required for the proper localization of Atg8 at the PAS under growing conditions. On the other hand, these mutant strains did not show any difference in GFP-Atg8 localization compared with the wild type strain under starvation conditions; most of the cells displayed GFP staining in their vacuoles, and about 75–90% of the cells showed a punctate dot of GFP-Atg8 at the PAS (Fig. 2). This result was consistent with previous observations that Ape1, Atg19, and Atg11 are dispensable for autophagy (8Scott S.V. Guan J. Hutchins M.U. Kim J. Klionsky D.J. Mol. Cell. 2001; 7: 1131-1141Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, 11Suzuki K. Kamada Y. Ohsumi Y. Dev. Cell. 2002; 3: 815-824Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 20Kim J. Kamada Y. Stromhaug P.E. Guan J. Hefner-Gravink A. Baba M. Scott S.V. Ohsumi Y. Dunn Jr., W.A. Klionsky D.J. J. Cell Biol. 2001; 153: 381-396Crossref PubMed Scopus (218) Google Scholar) (Fig. 1) and suggested that the PAS localization of Atg8 occurred independently of the recognition of the Cvt cargo under starvation conditions. It was reported that the PAS localization of Atg8 is dependent on its lipidation state (12Suzuki K. Kirisako T. Kamada Y. Mizushima N. Noda T. Ohsumi Y. EMBO J. 2001; 20: 5971-5981Crossref PubMed Scopus (806) Google Scholar). Therefore, we examined the lipidation of Atg8 in ape1Δ, atg19Δ, and atg11Δ cells and found there was no difference in lipidation between the wild type and these mutant strains (data not shown), suggesting that Atg8 still localized on some membrane structure, although it was not concentrated at the PAS in these mutant cells. Because Atg8 physically interacts with Atg19 (9Shintani T. Huang W.-P. Stromhaug P.E. Klionsky D.J. Dev. Cell. 2002; 3: 825-837Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar), we wondered whether only Atg8 lost its localization at the PAS or whether the overall organization of the PAS was compromised in the absence of the Cvt cargo. Recently, it was reported that the PAS localization of Atg2/Apg2 is important for its function in Cvt vesicle/autophagosome formation (13Shintani 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, 22Wang C.-W. Kim J. Huang W.-P. Abeliovich 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). Because its localization at the PAS is independent of Atg8, we selected Atg2 as a second PAS marker to examine the organization of the PAS. In rich medium, about 40% of wild type cells sh
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