Toxoplasma gondii Rab6 Mediates a Retrograde Pathway for Sorting of Constitutively Secreted Proteins to the Golgi Complex

Abstract

Toxoplasma gondii relies on protein secretion from specialized organelles for invasion of host cells and establishment of a parasitophorous vacuole. We identifyT. gondii Rab6 as a regulator of protein transport between post-Golgi dense granule organelles and the Golgi.Toxoplasma Rab6 was localized to cisternal rims of the late Golgi and trans-Golgi network, associated transport vesicles, and microdomains of dense granule and endosomal membranes. Overexpression of wild-type Rab6 or GTP-activated Rab6(Q70L) rerouted soluble dense granule secretory proteins to the Golgi and endoplasmic reticulum and augmented the effect of brefeldin A on Golgi resorption to the endoplasmic reticulum. Parasites expressing a nucleotide-free (Rab6(N124I)) or a GDP-bound (Rab6(T25N)) mutant accumulated dense granule proteins in the Golgi and associated transport vesicles and displayed reduced secretion of GRA4 and a delay in glycosylation of GRA2. Activated Rab6 on Golgi membranes colocalized with centrin during mitosis, and parasite clones expressing Rab6 mutants displayed a partial shift in cytokinesis from endodyogeny (formation of two daughter cells) to endopolygeny (multiple daughter cells). We propose that Toxoplasma Rab6 regulates retrograde transport from post-Golgi secretory granules to the parasite Golgi. Toxoplasma gondii relies on protein secretion from specialized organelles for invasion of host cells and establishment of a parasitophorous vacuole. We identifyT. gondii Rab6 as a regulator of protein transport between post-Golgi dense granule organelles and the Golgi.Toxoplasma Rab6 was localized to cisternal rims of the late Golgi and trans-Golgi network, associated transport vesicles, and microdomains of dense granule and endosomal membranes. Overexpression of wild-type Rab6 or GTP-activated Rab6(Q70L) rerouted soluble dense granule secretory proteins to the Golgi and endoplasmic reticulum and augmented the effect of brefeldin A on Golgi resorption to the endoplasmic reticulum. Parasites expressing a nucleotide-free (Rab6(N124I)) or a GDP-bound (Rab6(T25N)) mutant accumulated dense granule proteins in the Golgi and associated transport vesicles and displayed reduced secretion of GRA4 and a delay in glycosylation of GRA2. Activated Rab6 on Golgi membranes colocalized with centrin during mitosis, and parasite clones expressing Rab6 mutants displayed a partial shift in cytokinesis from endodyogeny (formation of two daughter cells) to endopolygeny (multiple daughter cells). We propose that Toxoplasma Rab6 regulates retrograde transport from post-Golgi secretory granules to the parasite Golgi. The Golgi complex coordinates secretory protein maturation and sorting and is a central intermediary of bidirectional transport between exocytic and endocytic pathways. In most protozoa, the early secretory pathway is well conserved in that of other eukaryotes, performing the critical function of biosynthetic transport to organelles, the cell surface, and the extracellular environment. Members of Apicomplexa, a diverse phylum of obligate intracellular parasites, are distinct from other eukaryotes in harboring three unique polarized secretory organelles, termed micronemes, rhoptries, and dense granules. As exemplified inToxoplasma gondii, sequential secretion from these organelles is essential for host cell invasion and the concomitant formation of an intracellular parasitophorous vacuole (PV) 1The abbreviations used are: PV, parasitophorous vacuole; TGN, trans-Golgi network; ER, endoplasmic reticulum; ARF1, ADP-ribosylation factor-1; SNARE, soluble underln]N-ethylmaleimide-sensitive fusion protein attachment protein receptor; RACE, rapid amplification of cDNA ends; ORF, open reading frame; HA, hemagglutinin; BAP, bacterial alkaline phosphatase; IMC, inner membrane complex; mAb, monoclonal antibody; CHO, Chinese hamster ovary; BFA, brefeldin A 1The abbreviations used are: PV, parasitophorous vacuole; TGN, trans-Golgi network; ER, endoplasmic reticulum; ARF1, ADP-ribosylation factor-1; SNARE, soluble underln]N-ethylmaleimide-sensitive fusion protein attachment protein receptor; RACE, rapid amplification of cDNA ends; ORF, open reading frame; HA, hemagglutinin; BAP, bacterial alkaline phosphatase; IMC, inner membrane complex; mAb, monoclonal antibody; CHO, Chinese hamster ovary; BFA, brefeldin A enveloping the parasite (1Joiner K.A. Roos D.S. J. Cell Biol. 2002; 157: 557-563Crossref PubMed Scopus (115) Google Scholar). Micronemes and rhoptries are apically tethered and secreted during attachment and penetration of a host cell. Dense granules inToxoplasma secrete aggregates of soluble and transmembrane proteins constitutively at a basal level and are stimulated for enhanced release following parasite invasion, suggesting a regulated, but Ca2+-independent, component (2Chaturvedi S. Qi H. Coleman D. Rodriguez A. Hanson P.I. Striepen B. Roos D.S. Joiner K.A. J. Biol. Chem. 1999; 274: 2424-2431Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Immunohistochemical analysis indicates that the sorting of dense granule proteins from those targeted to micronemes and rhoptries occurs at the late Golgi cisternae or trans-Golgi network (TGN). Although rhoptries originate from a precursor organelle formed by the Golgi during parasite division, the process regulating biogenesis ofToxoplasma dense granules is completely unknown. Two hypotheses are proposed to account for the biogenesis of regulated secretory vesicles in specialized mammalian cells (reviewed in Ref. 3Tooze S.A. Biochim. Biophys. Acta. 1998; 1404: 231-244Crossref PubMed Scopus (185) Google Scholar). In the selective aggregation model, secretory proteins aggregate in the TGN and are sorted for entry into secretory granules by receptors in the TGN membrane. Proteins that fail to aggregate may exit the TGN independently in constitutive secretory vesicles. In the sorting-by-retention model, regulated proteins are retained and sorted from constitutive secretory cargo after formation of immature secretory granules. In this model, immature secretory granules budding from the TGN contain the bulk of biosynthetic cargo and undergo maturation by selective budding of constitutive secretory vesicles containing non-aggregate proteins. Toxoplasma dense granule protein aggregates may dissociate and insert post-translationally into target membranes only after secretion into the PV (4Lecordier L. Mercier C. Sibley L.D. Cesbron-Delauw M.F. Mol. Biol. Cell. 1999; 10: 1277-1287Crossref PubMed Scopus (96) Google Scholar, 5Labruyere E. Lingnau M. Mercier C. Sibley L.D. Mol. Biochem. Parasitol. 1999; 102: 311-324Crossref PubMed Scopus (96) Google Scholar, 6Mercier C. Cesbron-Delauw M.F. Sibley L.D. J. Cell Sci. 1998; 111: 2171-2180PubMed Google Scholar). Understanding the biogenesis of Toxoplasma dense granules and the mechanisms involved in regulating dense granule protein transport will provide fundamental insight into the minimal requirements for sorting between regulated and constitutive secretory pathways in eukaryotes. Molecular evidence and genomic sequencing efforts indicate that the molecular machinery regulating vesicular transport in Apicomplexa is partially conserved in other eukaryotes, particularly early in the secretory pathway. Morphologically, these protozoa possess an endoplasmic reticulum (ER) that is contiguous with the nuclear envelope and a Golgi apparatus that ranges from single dispersed cisternae in malarial parasites (genus Plasmodium) to a stacked apically oriented Golgi in Toxoplasma. Components of the vesicle budding, transport, and fusion machinery have been cloned inToxoplasma, including N-ethylmaleimide-sensitive fusion protein, multiple Rab proteins (7Stedman T.T. Joiner K.A. Gordon S. Phagocytosis and Pathogens. 6. JAI Press Inc., Greenwich, CT1999: 233-261Google Scholar), ARF1 (8Liendo A. Stedman T.T. Ngo H.M. Chaturvedi S. Hoppe H.C. Joiner K.A. J. Biol. Chem. 2001; 276: 18272-18281Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar), and subunits of the coatomer (9Hager K.M. Striepen B. Tilney L.G. Roos D.S. J. Cell Sci. 1999; 112: 2631-2638Crossref PubMed Google Scholar) and adapter complexes (10Hoppe H.C. Ngo H.M. Yang M. Joiner K.A. Nat. Cell. Biol. 2000; 2: 449-456Crossref PubMed Scopus (116) Google Scholar). Evolutionarily conserved sorting motifs function in the transport of proteins to rhoptries and micronemes (10Hoppe H.C. Ngo H.M. Yang M. Joiner K.A. Nat. Cell. Biol. 2000; 2: 449-456Crossref PubMed Scopus (116) Google Scholar, 11Di Cristina M. Spaccapelo R. Soldati D. Bistoni F. Crisanti A. Mol. Cell. Biol. 2000; 20: 7332-7341Crossref PubMed Scopus (85) Google Scholar). In contrast, targeting motifs have not been found in dense granule proteins, and heterologous soluble proteins expressed with an N-terminal signal sequence are constitutively secreted into the PV through dense granules (12Karsten V. Qi H. Beckers C.J. Reddy A. Dubremetz J.F. Webster P. Joiner K.A. J. Cell Biol. 1998; 141: 1323-1333Crossref PubMed Scopus (111) Google Scholar). To further define regulation of dense granule secretion, we sought to identify molecular effectors of dense granule protein transport. In conjunction with effector proteins, monomeric GTPases of the Rab family localize to distinct intracellular compartments and confer one level of specificity on vesicle transport and fusion by mediating tethering or docking of membranes to of The Rab6 to function in transport A. J. B. M. B. J. Cell Biol. PubMed Scopus Google Scholar, C. J. B. Sci. S. A. PubMed Scopus Google Scholar) and retrograde transport from the Golgi to the in mammalian cells A. B. L. B. R. Nat. Cell. Biol. 1999; PubMed Scopus Google Scholar, J. L. F. A. S. S. P. B. A. B. J. Cell Biol. 1999; PubMed Scopus Google Scholar). The of was to function in early Golgi transport B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, B. 1998; PubMed Scopus Google Scholar). evidence indicates that of vesicles with late Golgi membranes and is a and the protein complex S. J. 2001; 20: PubMed Scopus Google Scholar, S. J. 2000; PubMed Scopus Google Scholar, Mol. Biol. Cell. 2001; PubMed Scopus Google Scholar). In this we that Toxoplasma Rab6 regulates protein transport at a between dense granules and the late Golgi cisternae and that or of Rab6 function partially constitutive secretion of proteins by transport to dense granules. The effect of Rab6 to the brefeldin resorption of constitutively secreted cargo and Golgi cisternae to the of GTP-activated or Rab6 mutants parasite to a partial in daughter parasite budding to that Toxoplasma Rab6 a retrograde pathway from post-Golgi secretory organelles to the late Golgi and may provide a between Rab6 function and and gondii cells in by of cell as D.S. Cell Biol. PubMed Scopus Google Scholar). gondii cells through the of D. B. Roos D.S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). gondii was with cDNA was with and amplification of cDNA was with Molecular and targeting conserved Rab proteins in the conserved or Rab6 proteins in and as for for and for for of from the used as for with the for was by with and with and used for with and the was with and the cloned into and by analysis of clones the and gondii including an of The open reading of gondii was from cDNA the the and of clones the of the to the and and Rab6 was by The gondii cDNA sequence been in the The of the used in the of cDNA are as of are in and is and analysis was after of of on and in and to membranes in and as J. Molecular A Scholar). A cDNA by and was and by with and the to a of in to Rab6 the was the analysis analysis was with The from analysis was to the single from the was of A gondii was by the The was into the of a D. PubMed Scopus Google Scholar), by of the signal sequence for protein and by amplification of with and cloned by into the of by of the with by amplification bacterial a was from and into and into the after amplification of with in was for of the and of from was for with or the heterologous secretory alkaline expressed from the and to the signal sequence (12Karsten V. Qi H. Beckers C.J. Reddy A. Dubremetz J.F. Webster P. Joiner K.A. J. Cell Biol. 1998; 141: 1323-1333Crossref PubMed Scopus (111) Google Scholar). from and by of the fusion in the Parasites by to D.S. Cell Biol. PubMed Scopus Google Scholar). of parasites in with and and cloned by on D.S. Cell Biol. PubMed Scopus Google Scholar). and from and from gondii and cells to and for or by to membranes. analysis was with the as with a gondii and on with containing gondii and proteins for as Sci. S. A. PubMed Scopus Google Scholar), that was for in the and in with for was to a of and for and the with of for and at on cell on with or gondii cells for at for as (12Karsten V. Qi H. Beckers C.J. Reddy A. Dubremetz J.F. Webster P. Joiner K.A. J. Cell Biol. 1998; 141: 1323-1333Crossref PubMed Scopus (111) Google Scholar). monoclonal of of and and A. A. B. D. Dubremetz J.F. PubMed Scopus Google Scholar). and and on a and cell with in from the for Cell was with by or and protein J. of The with with and and with a cell in and for for by the for Cell on with and and as three with minimal essential and in of minimal essential containing and of analysis of and secretion, parasites for analysis of parasites for and after three for and Parasites from host cells by in of containing (2Chaturvedi S. Qi H. Coleman D. Rodriguez A. Hanson P.I. Striepen B. Roos D.S. Joiner K.A. J. Biol. Chem. 1999; 274: 2424-2431Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) and at for at Cell in J. Molecular A Scholar) containing Molecular following the gondii soluble secreted proteins of the used for after of to as V. Qi H. Beckers C.J. Joiner K.A. Scopus Google Scholar) with to BAP, GRA4 or as and to and Rab6 in Golgi transport in and mammalian we sought to identify this protein inToxoplasma as a target for secretory protein amplification was used to a gondii In genomic a cDNA to a single gondii in is a in analysis a of with a In the to from host cell that Rab6 is of parasite The Toxoplasma cDNA a protein with a molecular of the the Toxoplasma Rab6 protein is most to Rab6 of the Apicomplexa malarial parasites and and The of gondii Rab6 to other Rab6 was by and analysis the The Rab family are well including the and confer and with Rab effectors and Rab6 a a essential for with a Rab6 A. F. P. J. B. Mol. Biol. Cell. 2000; PubMed Scopus Google Scholar). The and of in specificity and intracellular between Rab proteins, are Rab6 A single most well by was with of and Rab6 To a analysis of gondii we Toxoplasma Rab6 and Rab6 mutants to in N-terminal was to or to mutants Rab6(Q70L) and Rab6(Q70L) is to in is to for to protein F. PubMed Scopus Google Scholar, A. P. A. C. PubMed Scopus Google Scholar). bacterial by with to a gondii or with to the not in proteins with an molecular of was for and and but not of the of the In or and proteins as by to a to the parasite signal was in the parasite and in one or dense granules A and We that Rab6 motifs for Golgi conserved eukaryotes, the to of Rab proteins between and To the of Toxoplasma Rab6 a gondii Rab6 was expressed in cells and localized with including the tethering proteins and the protein and protein, a of late In to a gondii localized in a of the Golgi and and colocalized in this with signal on the Golgi In contrast, minimal with the and not To define Rab6 in parasite clones to Activated Rab6 was most on Golgi cisternae A Rab6 was on the of the late Golgi cisternae and the TGN, on the cisternal rims of the and and on vesicles associated with the cisternal of the the between the Golgi and the nuclear and the of the nuclear envelope was Rab6 microdomains on membranes of a of dense granule and Rab6 was associated with microdomains on membranes of vesicles, of endosomal but was not associated with membranes of rhoptries, micronemes, or A and Rab6 in a transport process to the late Golgi in dense granules. of Toxoplasma Rab6 in the parasite and cells not was to the brefeldin A an of the ARF1 of BFA, expressed to the with a and the parasite and Activated was dispersed by to a and suggesting a of the parasite Golgi into the ER, to retrograde in mammalian In these indicate Rab6 conserved of Rab6 proteins. To the ofToxoplasma Rab6 in transport and secretion of dense granule proteins, or with the secretory protein protein, to the signal as a soluble secreted dense granule protein in Toxoplasma (12Karsten V. Qi H. Beckers C.J. Reddy A. Dubremetz J.F. Webster P. Joiner K.A. J. Cell Biol. 1998; 141: 1323-1333Crossref PubMed Scopus (111) Google Scholar). localized to dense granules and the PV secretion and In the of or secretion into the PV and in dense granules partially with signal in the Golgi and the Rab6 augmented the retrograde of in the parasite the of The of and Rab6 mutants in secretory transport was by in GTP-activated mutant a but of in the Golgi and the envelope in with wild-type was in was partially of these mutants of the parasite in a vesicular and of dense granules with at or the late Golgi in the of secreted was in parasite of was parasite of parasite not transport of dense granule proteins and in parasites was and accumulated the Golgi in partially accumulated the Golgi with reduced dense granule of and in the of the Golgi and after secretion into the PV the is a for dense granule proteins in parasites with or Parasites expressing Rab6 mutants further for transport of dense granule proteins In the of accumulated in the with Although was dispersed from dense these for secreted proteins in the PV the parasites with suggesting an effect on transport of dense granule protein aggregates in with soluble secretory In contrast, transport of proteins to the cell and or proteins from the and Golgi was in the of the Rab6 mutants not these indicate that of Rab6 function by to partial in the transport of constitutively secreted proteins between dense granules and the Golgi. To bulk secretion into the PV was by Rab6 secretion of was in intracellular parasites by secreted from parasites or was of that secreted from parasites expressing of reduced secretion by the in of dense granules. that constitutive secretion may not transport through dense this is the in wild-type parasites (2Chaturvedi S. Qi H. Coleman D. Rodriguez A. Hanson P.I. Striepen B. Roos D.S. Joiner K.A. J. Biol. Chem. 1999; 274: 2424-2431Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, V. Qi H. Beckers C.J. Reddy A. Dubremetz J.F. Webster P. Joiner K.A. J. Cell Biol. 1998; 141: 1323-1333Crossref PubMed Scopus (111) Google Scholar). We the effect of level of Rab6 mutants in by parasite clones or with clones retained Rab6 with a single of the mutant proteins expressed from the level not In to parasites secretion was in parasite clones a secretion of with those expressing the mutant To these for dense granule proteins, we the secretion of GRA4 by in parasite clones expressing or the Rab6 mutant effect on dense granule secretion with parasites, of the mutant was secretion of GRA4 To the that protein in the parasite Golgi in the of the Rab6 mutants to retrograde of glycosylation the glycosylation of was by of is by glycosylation from an precursor to an protein A. A. B. D. Dubremetz J.F. PubMed Scopus Google Scholar, Striepen B. S. Dubremetz J.F. Mol. Biochem. Parasitol. 1998; PubMed Scopus Google Scholar). of a was in the of in parasites expressing in parasites expressing was with wild-type parasites of of was in parasite clones In of wild-type and GTP-activated Rab6 constitutive protein secretion, GDP-bound and nucleotide-free Rab6 mutants to dense granule protein transport and Rab6 to a retrograde pathway transport of dense granule proteins to the Golgi. and or of and Rab6 clones was reduced in with wild-type parasites Although Rab6 mutants not transport of proteins to the post-Golgi and parasites in precursor organelles not biogenesis is regulated, we the parasite clones for in the parasite by a process of budding termed process is through a of the and as a Roos D.S. Tilney L.G. J. Cell Sci. 2000; Google Scholar). mitosis, the Golgi in two the was in parasites expressing The of the Golgi in with the and to to nuclear Rab6 and centrin colocalized with suggesting a tethering of the late Golgi with the mutants are in and Rab6 with centrin during of a parasite in late with three daughter parasites and rhoptries an analysis of wild-type parasites endodyogeny with In daughter the as the of and and of endopolygeny in Toxoplasma and of with parasites endopolygeny two daughter is by and expressed as a of with daughter with parasites for parasites endopolygeny with of and and In one a daughter parasite is from the Golgi membranes with Rab6(Q70L) parasite and during cytokinesis with is with Golgi membranes during but only colocalized during daughter cell budding with In the a daughter is by a with with Golgi a for daughter cell is formed from and an associated network of the the and nuclear the into the two and parasite of division, multiple of by endopolygeny The of daughter a of organelles, including and that and with nuclear The of endopolygeny was to an of the in and daughter parasites, as a of daughter cell formation by Striepen B. Beckers C.J. Roos D.S. Mol. Biol. Cell. 2002; PubMed Scopus Google Scholar). In parasites by with two on of containing multiple daughter parasites found in a single in not and harboring parasites by endopolygeny and that Rab6 function of cytokinesis and in The polarized secretory pathway of gondii, with a single of apically oriented Golgi a for protein transport in eukaryotes. We have the gondii of the conserved Rab6 as the of the parasite Golgi and as a of retrograde transport from dense granule secretory Rab6 function in retrograde transport from and transport pathways in mammalian and in of Golgi proteins from the endosomal in Toxoplasma Rab6 to in post-Golgi transport of constitutively secreted proteins to the parasite Golgi. further that Rab6 function may for cytokinesis or that parasite cell may to of the retrograde pathway. We propose that the of Rab6 in Toxoplasma dense granule protein transport is retrograde from a post-Golgi of Rab proteins on the target membrane for vesicle transport. wild-type and GTP-activated Rab6 localize to the late Golgi cisternae and constitutive secretion of dense granule proteins only in Golgi and of the dense granule proteins. In this Rab6 the effect of on retrograde transport to the ER, in resorption of the Golgi cisternae and associated proteins mediating ARF1 that is essential for vesicle budding Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). Toxoplasma ARF1 mutants a in the early transport of dense granule proteins (8Liendo A. Stedman T.T. Ngo H.M. Chaturvedi S. Hoppe H.C. Joiner K.A. J. Biol. Chem. 2001; 276: 18272-18281Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar), that and pathways function in dense granule protein transport. In to and post-Golgi transport of dense granule proteins, these proteins in the Golgi and glycosylation of an in Golgi the of the mutants and the on constitutive secretion in clones indicate that Rab6 function is for secretion in in with in the of Rab6 with dense granule membranes and vesicles of the Golgi in to the of dense granule proteins in the Golgi by a transport pathway the two pathway may a function from dense granules or a in dense granule biogenesis or maturation through protein A retrograde transport in mammalian cells was proposed the of Rab6 on Golgi C. J. B. Sci. S. A. PubMed Scopus Google Scholar). pathway was further by following transport of and to the A. B. L. B. R. Nat. Cell. Biol. 1999; PubMed Scopus Google Scholar) and the that between the Golgi and at the mammalian cell J. L. F. A. S. S. P. B. A. B. J. Cell Biol. 1999; PubMed Scopus Google Scholar). is that this retrograde pathway may of proteins of and the of Golgi proteins. 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J. 2000; PubMed Scopus Google Scholar). the complex to the Golgi in the Golgi on vesicles and fusion S. J. 2001; 20: PubMed Scopus Google Scholar). is that an endosomal may between dense granules and the Golgi. Toxoplasma to a but distinct Golgi and mutants formation B. Stedman T.T. Ngo H.M. M. Joiner K.A. Cell 2002; PubMed Scopus Google Scholar). The transport of vesicles to the and in to transport M. D. D. Biochem. 2000; PubMed Google Scholar). Toxoplasma and Rab6 may as in pathways. of endosomal and compartments in Toxoplasma with and molecular in to A for Rab6 function was by the of Rab6 mutant parasites in budding is the of cytokinesis in Although and cytokinesis inToxoplasma as through the of Roos D.S. Tilney L.G. J. Cell Sci. 2000; Google Scholar), and nuclear are through with a of occurs as a of multiple nuclear and with complex and formation in the of the cell. from a of in Apicomplexa in multiple daughter are the cell cytokinesis the as a of daughter and parasite clones partially to Rab6 on Golgi membranes colocalized with centrin during suggesting a for Rab6 in cytokinesis with the nuclear effectors of Rab6 have been that may function in A and was through a Rab6 protein A. F. A. B. 1998; PubMed Scopus Google Scholar). protein with the effector of GTP-activated Rab6 and to in and in Rab6 may the and of with the as a of Golgi membranes and transport vesicles A. F. A. B. 1998; PubMed Scopus Google Scholar). is proposed to function in cell during cytokinesis E. M. J. 2000; PubMed Scopus Google Scholar, B. A. F. J. H. Mol. Cell. 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