The Ccz1-Mon1 Protein Complex Is Required for the Late Step of Multiple Vacuole Delivery Pathways

Mon1 and Ccz1 were identified from a gene deletion library as mutants defective in the vacuolar import of aminopeptidase I (Ape1) via the cytoplasm to vacuole targeting (Cvt) pathway. The mon1Δ and ccz1Δ strains also displayed defects in autophagy and pexophagy, degradative pathways that share protein machinery and mechanistic features with the biosynthetic Cvt pathway. Further analyses indicated that Mon1, like Ccz1, was required in nearly all membrane-trafficking pathways where the vacuole represented the terminal acceptor compartment. Accordingly, both deletion strains had kinetic defects in the biosynthetic delivery of resident vacuolar hydrolases through the CPY, ALP, and MVB pathways. Biochemical and microscopy studies suggested that Mon1 and Ccz1 functioned after transport vesicle formation but before (or at) the fusion step with the vacuole. Thus, ccz1Δ andmon1Δ are the first mutants identified in screens for the Cvt and Apg pathways that accumulate precursor Ape1 within completed cytosolic vesicles. Subcellular fractionation and co-immunoprecipitation experiments confirm that Mon1 and Ccz1 physically interact as a stable protein complex termed the Ccz1-Mon1 complex. Microscopy of Ccz1 and Mon1 tagged with a fluorescent marker indicated that the Ccz1-Mon1 complex peripherally associated with a perivacuolar compartment and may attach to the vacuole membrane in agreement with their proposed function in fusion. Mon1 and Ccz1 were identified from a gene deletion library as mutants defective in the vacuolar import of aminopeptidase I (Ape1) via the cytoplasm to vacuole targeting (Cvt) pathway. The mon1Δ and ccz1Δ strains also displayed defects in autophagy and pexophagy, degradative pathways that share protein machinery and mechanistic features with the biosynthetic Cvt pathway. Further analyses indicated that Mon1, like Ccz1, was required in nearly all membrane-trafficking pathways where the vacuole represented the terminal acceptor compartment. Accordingly, both deletion strains had kinetic defects in the biosynthetic delivery of resident vacuolar hydrolases through the CPY, ALP, and MVB pathways. Biochemical and microscopy studies suggested that Mon1 and Ccz1 functioned after transport vesicle formation but before (or at) the fusion step with the vacuole. Thus, ccz1Δ andmon1Δ are the first mutants identified in screens for the Cvt and Apg pathways that accumulate precursor Ape1 within completed cytosolic vesicles. Subcellular fractionation and co-immunoprecipitation experiments confirm that Mon1 and Ccz1 physically interact as a stable protein complex termed the Ccz1-Mon1 complex. Microscopy of Ccz1 and Mon1 tagged with a fluorescent marker indicated that the Ccz1-Mon1 complex peripherally associated with a perivacuolar compartment and may attach to the vacuole membrane in agreement with their proposed function in fusion. Compartmentalization allows eukaryotic cells to regulate intracellular functions by separating competing reactions and localizing enzymes and substrates at specific locations within the cell. Efficient compartmentalization necessitates dynamic protein trafficking processes by which cells are able to establish and maintain the identity and function of each organelle. The vacuole (lysosome) of the yeast Saccharomyces cerevisiae plays a central role in the turnover of cytoplasmic organelles, degradation of intracellular/extracellular components, and maintenance of cellular physiology (1Klionsky D.J. Herman P.K. Emr S.D. Microbiol. Rev. 1990; 54: 266-292Google Scholar). To carry out these functions, the vacuole maintains a variety of degradative enzymes. Both resident hydrolases and their substrates arrive at this destination through a variety of sorting pathways. The main routes by which vacuolar hydrolases are delivered to this organelle are the carboxypeptidase Y (CPY), 1The abbreviations used are: CPY, carboxypeptidase Y; ALP, alkaline phosphatase; Ape1, aminopeptidase I; CFP, cyan fluorescent protein; Cvt, cytoplasm to vacuole targeting; GFP, green fluorescent protein; prApe1, precursor aminopeptidase I; PVC, pre-vacuolar compartment; SMD, synthetic minimal medium with dextrose; SD/-N, synthetic minimal medium with dextrose but lacking nitrogen; YFP, yellow fluorescent protein; ORF, open reading frame; MES, 4-morpholineethanesulfonic acid; PIPES, 1,4-piperazinediethanesulfonic acid alkaline phosphatase (ALP), and multivesicular body (MVB) pathways, which involve transit through a portion of the secretory pathway, and the cytoplasm to vacuole targeting (Cvt) pathway by which the cargo molecules are packaged as cytosolic membrane-bound intermediates (2Kim J. Scott S.V. Klionsky D.J. Int. Rev. Cytol. 2000; 198: 153-201Google Scholar, 3Lemmon S.K. Traub L.M. Curr. Opin. Cell Biol. 2000; 12: 457-466Google Scholar). Resident proteins are also transmitted by inheritance from mother cell vacuoles to daughter cells during cell division (4Weisman L.S. Wickner W. Science. 1988; 241: 589-591Google Scholar). Substrates enter the vacuole through endocytosis, autophagy and the vacuole import and degradation pathway (reviewed in Ref. 5Klionsky D.J. Ohsumi Y. Annu. Rev. Cell Dev. Biol. 1999; 15: 1-32Google Scholar). One common feature in all of these processes is membrane fusion. The membrane fusion mechanism acts to ensure specificity for the directed movement of proteins while also maintaining the distinct composition of each organelle within the highly compartmentalized eukaryotic cell. The cytoplasm to vacuole targeting pathway that is used to deliver the soluble hydrolase aminopeptidase I (Ape1) to the vacuole has been under investigation (for reviews see Refs. 2Kim J. Scott S.V. Klionsky D.J. Int. Rev. Cytol. 2000; 198: 153-201Google Scholar, 5Klionsky D.J. Ohsumi Y. Annu. Rev. Cell Dev. Biol. 1999; 15: 1-32Google Scholar, and 6Klionsky D.J. J. Biol. Chem. 1998; 273: 10807-10810Google Scholar). Under vegetative conditions, precursor Ape1 (prApe1) is assembled into a large Cvt complex composed in part of multiple prApe1 dodecamers in the cytosol that becomes enwrapped within a double-membrane Cvt vesicle (7Baba M. Osumi M. Scott S.V. Klionsky D.J. Ohsumi Y. J. Cell Biol. 1997; 139: 1687-1695Google Scholar). Upon completion, the cytosolic Cvt vesicle targets to the vacuole. The outer membrane of the Cvt vesicle fuses with the vacuole membrane and the intact inner vesicle (Cvt body) passes into the vacuole lumen (8Scott S.V. Baba M. Ohsumi Y. Klionsky D.J. J. Cell Biol. 1997; 138: 37-44Google Scholar). The Cvt body is ultimately broken down by resident vacuolar hydrolases, resulting in the release and maturation of prApe1. Precursor Ape1 is transported to the vacuole by another pathway, termed autophagy (Apg), under (2Kim J. Scott S.V. Klionsky D.J. Int. Rev. Cytol. 2000; 198: 153-201Google Scholar, D.J. Emr S.D. Science. 2000; Scholar). the Apg pathway, of cytoplasm are within membrane that are also to the vacuole (7Baba M. Osumi M. Scott S.V. Klionsky D.J. Ohsumi Y. J. Cell Biol. 1997; 139: 1687-1695Google Scholar). Apg is a degradative mutants defective in with mutants M. Klionsky D.J. J. Biol. Chem. Scholar). and analyses that the Cvt and Apg pathways (2Kim J. Scott S.V. Klionsky D.J. Int. Rev. Cytol. 2000; 198: 153-201Google Scholar, 5Klionsky D.J. Ohsumi Y. Annu. Rev. Cell Dev. Biol. 1999; 15: 1-32Google Scholar, D.J. Emr S.D. Science. 2000; Scholar). To into the pathways, a gene deletion library for mutants that are defective in prApe1 mutants that are required for import that had been in these pathways. The of of these Ccz1, has been suggested to in multiple trafficking pathways to the vacuole J. J. Cell 2000; Scholar). of the protein the to and with the ccz1Δ The has been identified as a specific that the ccz1Δ J. J. Cell Scholar). the Ccz1 and J. J. Cell Scholar). The mon1Δ is to and M. but is this that strains lacking of these proteins Both Mon1 and Ccz1 are required for the pathways but also vacuole processes the sorting of vacuolar proteins through the CPY, ALP, and MVB pathways and Biochemical and that the pathways are at a after the formation of the but to their fusion with the vacuole. studies also that Ccz1 and Mon1 to a membrane and that physically the in of these proteins to a perivacuolar compartment and the vacuole a with their proposed role in fusion. The yeast strains used in this are in minimal medium yeast and and as medium yeast and and medium yeast and cerevisiae strains were at cells used for this were in the medium to of strains used in this Klionsky D.J. L.M. Emr S.D. Biol. 1988; S.V. L.S. Y. M. Ohsumi Y. Klionsky D.J. J. Biol. Chem. 2000; S.V. L.S. Y. M. Ohsumi Y. Klionsky D.J. J. Biol. Chem. 2000; Klionsky D.J. L.M. Emr S.D. Biol. 1988; Emr S.D. J. 1998; J. Scott S.V. Klionsky D.J. Biol. 1999; J. Biol. Chem. 1998; 273: in a for medium were from and alkaline phosphatase were from was from was from was from and was from The the marker was a from of The and were from J. 1998; Scholar). The and were from the of was from were from Ape1 D.J. J. Cell Biol. D.J. L.M. Emr S.D. Biol. 1988; and D.J. L.M. Emr S.D. Biol. 1988; been and were by of Wickner and of and were from and the was from To Mon1, the of the Mon1 was and to the of the The resulting was into protein and were as J. Klionsky D.J. J. Biol. Chem. Scholar). gene deletion library was from The mutants from the were and at for The cells were and in of MES, and and into cell by and The were to The and were by a J. Baba M. Ohsumi Y. Klionsky D.J. J. Cell Biol. 2000; Scholar). the marker was from the by that of the by that at the and of the were used to yeast strains were by for the Ape1 were for the and fluorescent protein To and both and their were as The resulting for before the the and after the The were with and into the of the to The for the of and of the The were with to To Ccz1, was as a The resulting was and into and that a D.J. Emr S.D. Cell Biol. 1999; Scholar). To fusion to Mon1, the was as a The resulting were into to The was with and into to with The J. Klionsky D.J. J. Biol. Chem. J. Biol. Chem. 1999; J. Klionsky D.J. J. Cell Biol. Emr S.D. J. 1997; and J. were and of the was out as Scott S.V. Klionsky D.J. J. Cell Biol. Scholar). kinetic of yeast cells were to of and into The from of cells were in of medium and with of for by a in yeast and at a of were at the indicated and was to the The were to a for The resulting and were with acid were in and to as Scott S.V. Klionsky D.J. J. Cell Biol. Scholar). kinetic analyses of Ape1, and yeast cells were to of in were in of medium and with of for by a as at a of were at the indicated and with were by and to as Scott S.V. Klionsky D.J. J. Cell Biol. Scholar). Cell and and degradation were as J. Klionsky D.J. J. Biol. Chem. Scholar). The membrane was by the J. Y. J. Baba M. Scott S.V. Ohsumi Y. Klionsky D.J. J. Cell Biol. with from the were in PIPES, at a of The was at for at The from of cells were in of in with the of The were with of in and with of in The resulting step was to at for at The was as the the was as the and the was as the The were with and by The was as J. Klionsky D.J. J. Biol. Chem. Scholar). were to in The were at for and the were in in the of were out for by acid and cells were to in The cells were into and in and the at a of a for at the was to for resulting in the and The was to for at to the and The resulting were to To membrane of and the membrane from were with as J. Klionsky D.J. J. Biol. Chem. Scholar). was a of a J. Klionsky D.J. J. Biol. Chem. Scholar). a was to and into The were to in and a and The was to to the intact this the from of cells was at for at The resulting membrane was in of and of a of in and a The were to at for at in a were from the of the into The were and with by The for co-immunoprecipitation with was from a J. J. Cell Scholar). of cells were with in with the of and a cell were at for at To the resulting of was by with protein at were with a of proteins were in by and strains used for microscopy were in medium to was as Emr S.D. J. Cell Biol. Scholar). Microscopy was a fluorescent were by mutants defective in the Cvt and Apg pathways been and (reviewed in Refs. 5Klionsky D.J. Ohsumi Y. Annu. Rev. Cell Dev. Biol. 1999; 15: 1-32Google and J. Klionsky D.J. Annu. Rev. 2000; these pathways to are in the mechanism the dynamic of the Cvt and Apg pathways. that the of mutants into the protein machinery of these Accordingly, a gene deletion library the of prApe1, a cargo protein that is delivered to the vacuole through the pathways. the mutants mon1Δ and ccz1Δ a in prApe1 mon1Δ has been as a role in the Cvt pathway, analyses that is a gene M. Klionsky D.J. J. Biol. Chem. Scholar). The ccz1Δ was identified to to J. 1999; 15: Scholar). has also been that the a vacuole that Ccz1 to the and has been suggested to in with the protein J. J. Cell 2000; Scholar, J. J. Cell Scholar). has been a Mon1 a acid protein with a of that Mon1 with proteins identity with Mon1 in and Ccz1 has cells are under conditions, the of Ape1 is as the a is as the both and ccz1Δ strains the precursor of The in prApe1 in these mutants was by of the the of Mon1 and Ccz1 for the Cvt pathway Precursor Ape1 is delivered to the vacuole through autophagy under a to the mon1Δ and ccz1Δ strains were able to carry out mutants specific to the Cvt pathway, are while mutants defective for autophagy in the of J. Klionsky D.J. J. Biol. Chem. Scholar). in the was to the mon1Δ to the displayed a of in in was these cells Mon1 from a that autophagy is in the mon1Δ and ccz1Δ that mutants that are autophagy defective by this are able to the formation of under is but is able to the formation of in J. Klionsky D.J. J. Cell Biol. 2000; Scholar). of the Cvt and Apg pathways are for of these pathways. and are required for the Cvt pathway to function in autophagy J. Y. J. Baba M. Scott S.V. Ohsumi Y. Klionsky D.J. J. Cell Biol. Scholar, S.V. L.S. Y. M. Ohsumi Y. Klionsky D.J. J. Biol. Chem. 2000; Scholar, Y. Ohsumi M. Ohsumi Y. J. Cell Biol. 2000; Scholar). Accordingly, these of mutants are able to prApe1 under of autophagy by the role of Ccz1 and Mon1 in prApe1 import under were in to to medium lacking and the of prApe1 was by the a of the prApe1 after cells were to the that is defective for both the Cvt and Apg pathways was to prApe1 to in to the the mon1Δ and ccz1Δ strains the precursor of Ape1 in conditions, that these proteins are required for The in prApe1 maturation in was with the Thus, that Mon1 and Ccz1 are required for both the Cvt and autophagy pathways. that the degradation pathway, pexophagy, as the Cvt and autophagy pathways M. Klionsky D.J. J. Cell 1999; Scholar). To Mon1 and Ccz1 are also required for pexophagy, the of by cells in acid in the and ccz1Δ and the degradation of after cells were to cell were at the indicated and by in degradation both the mon1Δ strains at the a in that both Mon1 and Ccz1 are part of the mechanism by the Cvt, and pathways. has been that the ccz1Δ a vacuolar hydrolase sorting as as a vacuole J. J. Cell 2000; Scholar). To a of vacuole protein delivery in the mon1Δ cargo proteins that are to the vacuole by is transported to the vacuole through the pathway, a transport that the and the is with a of of is from the cell under used for this of in mutants as as and the is into the as the the mon1Δ a of was in the intracellular the of the protein was in the after of and was to the were with The in the ccz1Δ has been J. J. Cell 2000; Scholar). with the a sorting by but a of under The of a in from a pre-vacuolar compartment that has J. Cell Biol. Scholar). the delivery of the vacuole membrane protein through the pathway. Under conditions, both and ccz1Δ strains of while the and Ref. J. J. Cell 2000; Scholar). To the delivery of in these the of used a as a the is required for the pathway. and strains were to and by fluorescent to the vacuole in the both ccz1Δ andmon1Δ also displayed a vacuole a of cells vacuoles and was at the vacuole membrane delivery of this hydrolase to the vacuole. to the in multiple in vacuoles in the are highly were able to that of these fluorescent were of the that both the ccz1Δ andmon1Δ strains the vacuole membrane but also displayed of their a in the delivery of were by cells which is also delivered to the vacuole by the pathway a in the pathway in the mon1Δ and ccz1Δ to the CPY, and pathways, proteins for the vacuole also transit through the and MVB pathways. by at the of in and ccz1Δ is and is by both and of J. Cell Biol. 2000; Scholar). this the the was in the vacuole was to multiple of the vacuole in and ccz1Δ may vesicles. in the mon1Δ and ccz1Δ the of through the MVB pathway J. Scholar). to the vacuole lumen in the in the ccz1Δ and mon1Δ cells displayed a large of of vacuoles which may the were MVB pathway marker proteins and The of mutants identified were specific to the Cvt and autophagy pathways and defects in vacuole delivery pathways. mutants all to function at the of vesicle the mutants were to and Scott S.V. Klionsky D.J. J. Cell Biol. a with a role in vacuole protein and ccz1Δ mutants are defective in multiple vacuole delivery pathways, that Mon1 and Ccz1 for protein trafficking pathways through their for the vesicle fusion step with the vacuole. To the proposed role of Ccz1 and Mon1 for the fusion of with the that the in the transport of prApe1 J. Klionsky D.J. J. Biol. Chem. Scholar). To prApe1 was able to a membrane from was to through a step a portion of prApe1 and the membrane protein were and into the in the of the cytosolic protein was in the was with the ccz1Δ that prApe1 is able to to To prApe1 is within completed Cvt out a were as under and the were to in the of is defective in the of to and is to completed Cvt M. Ohsumi M. Ohsumi Y. Biol. 1999; J. Scott S.V. Klionsky D.J. Biol. 1999; Scholar). prApe1 in a in the of is a protein that is required for the fusion of Cvt with the vacuole J. Scott S.V. Klionsky D.J. Biol. 1999; and cells accumulate prApe1. Precursor Ape1 in the mon1Δ strains was also in the of that within completed vesicles. The of into the that of prApe1 was to To prApe1 was within cytosolic of the Cvt pathway by at in is required for Cvt vesicle and formation and associated with these J. Klionsky D.J. J. Cell Biol. Scholar, Y. Y. Ohsumi M. Ohsumi Y. J. Cell Biol. 2000; Scholar). Thus, as a vesicle with was as a the vacuole in the cells in medium Under conditions, is and a vacuole lumen of in the in and ccz1Δ strains displayed multiple to that in cells and Ref. Y. Ohsumi Y. J. Scholar). the fluorescent and that the multiple in these strains were of the Under conditions, a in the strains that are defective in membrane that were of vacuoles in the strains but of the to with the vacuole. these that prApe1 is within completed cytosolic in both the mon1Δ Thus, that Ccz1 and Mon1 are required for the fusion step of these with the vacuole. to the of Ccz1 and Mon1, tagged both proteins with the The of Ccz1 Mon1, the the of cells and the vacuole has been that Ccz1 is in the and J. J. Cell 2000; Scholar). to with with tagged at the was with to and to by The were to as under The cytosolic protein was from the while the vacuole membrane protein was in the also the of and was in the was in the and that was in the and but that a also in the a cytosolic of this protein analyses for these proteins by the of their membrane that both and were from the membrane by with and while of each protein membrane in the of and these that and are peripherally to a membrane that are The of in the of may that both proteins with a large protein complex. To the of of Ccz1 and Mon1 in strains where was to the of the at the strains displayed a vacuolar that the fusion proteins are cells in to in minimal were was in perivacuolar cell and also displayed a vacuole membrane were and to the vacuoles had a to the fluorescent was yeast cells under vacuoles to and the and yeast were with to the vacuoles to The of and was and was by the vacuolar by the to SMD, were able to the with the vacuole of and The and within of the that the of Ccz1-Mon1 to membrane to the vacuole and attach to the vacuole membrane to their both proteins displayed a by fluorescent the by the of and Mon1 tagged at the with a fluorescent under the of the The resulting vacuolar and multiple to the with the the proteins The with and was from the with proteins that to the J. Klionsky D.J. J. Biol. Chem. Scholar). To Mon1 and Ccz1 to a distinct the of to is a for prApe1 S.V. J. J. Klionsky D.J. and to the J. Klionsky D.J. J. Biol. Chem. Scholar). that the to with the represented by that the ccz1Δ and mon1Δ strains vacuole protein transport and that Ccz1 and Mon1 are both and that their with has both proteins by fluorescent microscopy To the of and the membrane as under were from the of the and by and were both in through their with and these proteins displayed fractionation that were distinct from and also the of that has been suggested to interact with Ccz1, and that a of these proteins in and but that the were distinct To this Ccz1 physically with Mon1, a co-immunoprecipitation a of and in of the proteins for the strains with to prApe1 maturation were with and the cell was to as the Mon1 as under The a of in the and a of this in cells a and were to a with as under The were to and Mon1, and the the complex down a of the Mon1 The is specific Mon1 Mon1 was in the mon1Δ under the Mon1 was in the of of the Mon1 in the complex that the of Mon1 was Ccz1 The was also out the Mon1 has been that with J. J. Cell Scholar). were to in this is that the and Ccz1 also is the Ccz1 and Mon1 is and The biosynthetic Cvt pathway that the precursor of the vacuolar hydrolase Ape1 from the cytoplasm to the yeast vacuole is the of identified mutants and that of and and mutants that are defective in the degradative autophagy pathway (reviewed in Refs. 5Klionsky D.J. Ohsumi Y. Annu. Rev. Cell Dev. Biol. 1999; 15: 1-32Google and J. Klionsky D.J. Annu. Rev. 2000; Scholar). To a of the Cvt pathway, identified mutants defective in prApe1 sorting by the yeast deletion this identified Ccz1 and Mon1, required for the Cvt, autophagy and pathways To the of these proteins first to and mon1Δ strains had in vacuole transport pathways. The pathway transport through a portion of the secretory pathway, and the compartment that the pathway cargo protein was as the in has been for the ccz1Δ J. J. Cell 2000; Scholar). The pathway from the pathway in the cargo proteins as through the before the vacuole. a in in these strains the of of the vacuoles in these a large of that were the see vesicle in the ccz1Δ and mon1Δ the of to their vacuoles Thus, that the pathway that the is in the ccz1Δ andmon1Δ The of the vacuole resulting from the of and hydrolases that the pathway. is by the of precursor in vacuoles from the ccz1Δ andmon1Δ strains of and in the and MVB pathways that mon1Δ defects in multiple pathways. The in multiple vacuole delivery pathways and the of transport intermediates in the mutants suggested that Mon1 and Ccz1 at the of fusion with the vacuole. of the for the pathway J. Klionsky D.J. Annu. Rev. 2000; the role of Mon1 and Ccz1 through analyses that the of prApe1 J. Klionsky D.J. J. Biol. Chem. Scholar). the the pathway into vesicle and cargo vesicle and vesicle by maturation of prApe1 Accordingly, to the at which the cargo protein prApe1 during transport in The membrane indicated that prApe1 in the mon1Δ was that prApe1 in the mon1Δ and ccz1Δ strains was in a that the step for the Cvt complex was Thus, mon1Δ are the first mutants that been in screens for and that after of the vesicles. prApe1 accumulate in within the vacuole lumen in strains defective in the vesicle To that the ccz1Δ and are defective in delivery to the the of in the is a that is required for Cvt vesicle formation and is to Cvt Scott S.V. J. Klionsky D.J. J. Biol. Chem. 2000; Scholar). Accordingly, as a vesicle to the perivacuolar in the is of the vacuole in multiple in the ccz1Δ andmon1Δ strains the of in the mutants was to that in the is a that is required for the fusion of multiple vesicle Cvt and with the vacuole J. Scott S.V. Klionsky D.J. Biol. 1999; Scholar). that Ccz1 and Mon1 function at the of fusion of with the vacuole. Ccz1 and Mon1 are both membrane proteins is cytosolic of Ccz1 The proteins to a compartment that at the as Both proteins to function as a stable protein complex termed the Ccz1-Mon1 complex in this in of Ccz1 and Mon1 tagged with fluorescent suggested that the Ccz1-Mon1 complex to perivacuolar that The that Ccz1 with the marker J. J. Cell 2000; that these of protein to the that is to the of Cvt the of the membrane also vacuole membrane for both Mon1 and Ccz1, these proteins to the vacuole membrane to their function in fusion. a vacuole of Ccz1 Mon1 in out the that their with the vacuole is and is during the is with which is from the vacuole J. Klionsky D.J. Biol. 12: Scholar). of the of the in the pathways is in the fusion proteins Emr S.D. J. Cell Biol. 1997; 138: Biol. 1999; and Emr S.D. Biol. 1998; are required for both Cvt vesicle and fusion with the vacuole. the J. Scott S.V. Klionsky D.J. Biol. 1999; and proposed termed the fusion and vacuole protein complex that and Scott S.V. Klionsky D.J. J. Cell Biol. Scholar, Emr S.D. 2000; is also machinery at this these identified required for the Cvt fusion a vacuole are from the the but all mutants that a vacuole are defective for the pathway. the a vacuole the of Ape1 M. Wickner W. Biol. Scholar). and J. mutants as vacuoles but are for import of prApe1 D.J. J. Cell Biol. Scholar). this the Ccz1-Mon1 complex as at the fusion step in the pathways, as as in pathways that involve vesicle fusion with the vacuole. of role in vacuole and is to the Ccz1-Mon1 complex is part of the vacuole fusion is the specific role of the Ccz1-Mon1 Ccz1 has been to interact with J. J. Cell Scholar). the Ccz1-Mon1 complex also part of the co-immunoprecipitation the of and that this is the Ccz1-Mon1 complex is and are to the specific role of the Ccz1-Mon1 complex in the Cvt and Apg pathways. in into their function in the mechanism of vesicle fusion. Scott and Wickner and the for and of the Klionsky and for and