The AP-1 Clathrin-adaptor Is Required for Lysosomal Enzymes Sorting and Biogenesis of the Contractile Vacuole Complex in<i>Dictyostelium</i>Cells

Lefkir, Yaya; Chassey, Benoı̂t de; Dubois, Annick; Bogdanovic, Aleksandra; Brady, Rebecca J.; Destaing, Olivier; Brückert, Franz; O’Halloran, Theresa J.; Cosson, Pierre; Letourneur, François

doi:10.1091/mbc.e02-10-0627

Cited by 46 publications

(62 citation statements)

References 80 publications

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“…The interaction between ␥-adaptin and Rh50 is consistent with our previous study that demonstrated the role of AP-1 coat in the biogenesis of the CV (Lefkir et al, 2003). AP-1-coated vesicles could play an essential role in intracellular transport Journal of Cell Science 119 (5) Fig.…”

Section: Discussionsupporting

confidence: 91%

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Acidic clusters target transmembrane proteins to the contractile vacuole inDictyosteliumcells

Mercanti

Blanc

Lefkir

et al. 2006

Journal of Cell Science

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The mechanisms responsible for the targeting of transmembrane integral proteins to the contractile vacuole (CV) network in Dictyostelium discoideum are unknown. Here we show that the transfer of the cytoplasmic domain of a CV-resident protein (Rh50) to a reporter transmembrane protein (CsA) is sufficient to address the chimera (CsA-Rh50) to the CV. We identified two clusters of acidic residues responsible for this targeting, and these motifs interacted with the gamma-adaptin AP-1 subunit in a yeast protein-protein interaction assay. For the first time we report the existence of an indirect transport pathway from the plasma membrane to the CV via endosomes. Upon internalization, the small fraction of CsA-Rh50 present at the cell surface was first concentrated in endosomes distinct from early and late p80-positive endosomes and then slowly transported to the CV. Together our results suggest the existence of an AP-1-dependent selective transport to the contractile vacuole in Dictyostelium

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Section: Discussionsupporting

confidence: 91%

“…Deletion of the apm1 gene encoding the medium subunit of the clathrin adaptorprotein AP-1 also results in the absence of a characteristic CV (Lefkir et al, 2003). Together these results suggest that clathrin-coated vesicles, and more precisely AP-1 clathrincoated vesicles, are involved in the biogenesis of the CV.…”

Section: Introductionmentioning

confidence: 82%

Acidic clusters target transmembrane proteins to the contractile vacuole inDictyosteliumcells

Mercanti

Blanc

Lefkir

et al. 2006

Journal of Cell Science

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“…We also generally observed in the disruption lines that stalks and spores were thin and smaller than those of the parental Ax2 cells. Interestingly, similar observations were observed in the AP-1 clathrinadaptor protein null cell development in Dictyostelium (Lefkir et al, 2003). These results suggest that VwkA may have physiological functions that are different from those of MHCK A, MHCK B, and MHCK C during development of D. discoideum.…”

Section: Vwka Gene Disruption Defectssupporting

confidence: 81%

Identification and Characterization of a Novel α-Kinase with a von Willebrand Factor A-like Motif Localized to the Contractile Vacuole and Golgi Complex inDictyostelium discoideum

Betapudi

Mason

Licate

et al. 2005

MBoC

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We have identified a new protein kinase in Dictyostelium discoideum that carries the same conserved class of "␣-kinase" catalytic domain as reported previously in myosin heavy chain kinases (MHCKs) in this amoeba but that has a completely novel domain organization. The protein contains an N-terminal von Willebrand factor A (vWFA)-like motif and is therefore named VwkA. Manipulation of VwkA expression level via high copy number plasmids (VwkA ؉؉ cells) or gene disruption (vwkA null cells) results in an array of cellular defects, including impaired growth and multinucleation in suspension culture, impaired development, and alterations in myosin II abundance and assembly. Despite sequence similarity to MHCKs, the purified protein failed to phosphorylate myosin II in vitro. Autophosphorylation activity, however, was enhanced by calcium/calmodulin, and the enzyme can be precipitated from cellular lysates with calmodulin-agarose, suggesting that VwkA may directly bind calmodulin. VwkA is cytosolic in distribution but enriched on the membranes of the contractile vacuole and Golgi-like structures in the cell. We propose that VwkA likely acts indirectly to influence myosin II abundance and assembly behavior and possibly has broader roles than previously characterized ␣ kinases in this organism, which all seem to be MHCKs. INTRODUCTIONNearly all aspects of cell life are regulated by protein phosphorylation involving a large number of biochemical reactions and distinct signaling pathways. Existence of ϳ500 genes encoding various protein kinases in the human genome further underscores their importance in cell life (Manning et al., 2002). Most of these conventional protein kinases belong to serine/threonine/tyrosine, a kinase superfamily whose members carry a conserved catalytic domain with recognizable sequence similarity in spite of having different targets in the cell (Hardie, 1994;Hanks and Hunter, 1995). In recent years, however, biochemical and molecular approaches have led to the identification of more divergent classes of eukaryotic protein kinases carrying a catalytic domain sequence with no sequence similarity with conventional protein kinase catalytic domains (Manning et al., 2002). One major class of unconventional protein kinases was first identified via representatives of myosin heavy chain kinases (MHCKs) in Dictyostelium discoideum (Futey et al., 1995;Côté et al., 1997) and via identification of mammalian eEF-2 kinase (eEF-2K) as an unconventional protein kinase (Ryazanov et al., 1997). This novel, conserved group of unconventional eukaryotic protein kinases has been termed the ␣-kinase family (Ryazanov et al., 1999;Drennan and Ryazanov, 2004).Molecular cloning and biochemical characterization studies demonstrated that at least three Dictyostelium ␣-kinases genes encode enzymes that can specifically phosphorylate myosin II heavy chain (MHC) in vitro and in vivo, and were thus named as MHC kinase A (MHCK A), MHC kinase B (MHCK B) and MHC kinase C (MHCK C) (Futey et al., 1995;Clancy et al., 1997;Luo et al., 200...

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“…All Dictyostelium cells used in this study were derived from the subclone DH1-10 (Cornillon et al, 2000) of the DH1 strain (Caterina et al, 1994). The 1 (Lefkir et al, 2003) and myoVII (Gebbie et al, 2004) knockout strains have been described previously. The myoK (Schwarz et al, 2000) knockout cells were a gift from T. Soldati (University of Geneva, Switzerland).…”

Section: Cells and Reagentsmentioning

confidence: 99%

Selective membrane exclusion in phagocytic and macropinocytic cups

Mercanti

Charette

Bennett

et al. 2006

Journal of Cell Science

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Specialized eukaryotic cells can ingest large particles and sequester them within membrane-delimited phagosomes. Many studies have described the delivery of lysosomal proteins to the phagosome, but little is known about membrane sorting during the early stages of phagosome formation. Here we used Dictyostelium discoideum amoebae to analyze the membrane composition of newly formed phagosomes. The membrane delimiting the closing phagocytic cup was essentially derived from the plasma membrane, but a subgroup of proteins was specifically excluded. Interestingly the same phenomenon was observed during the formation of macropinosomes, suggesting that the same sorting mechanisms are at play during phagocytosis and macropinocytosis. Analysis of mutant strains revealed that clathrin-associated adaptor complexes AP-1, -2 and -3 were not necessary for this selective exclusion and, accordingly, ultrastructural analysis revealed no evidence for vesicular transport around phagocytic cups. Our results suggest the existence of a new, as yet uncharacterized, sorting mechanism in phagocytic and macropinocytic cups

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The AP-1 Clathrin-adaptor Is Required for Lysosomal Enzymes Sorting and Biogenesis of the Contractile Vacuole Complex inDictyosteliumCells

Cited by 46 publications

References 80 publications

Acidic clusters target transmembrane proteins to the contractile vacuole inDictyosteliumcells

Acidic clusters target transmembrane proteins to the contractile vacuole inDictyosteliumcells

Identification and Characterization of a Novel α-Kinase with a von Willebrand Factor A-like Motif Localized to the Contractile Vacuole and Golgi Complex inDictyostelium discoideum

Selective membrane exclusion in phagocytic and macropinocytic cups

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