The Golgi-localized, γ-ear-containing, ARF binding proteins (GGAs) are a highly conserved family of monomeric clathrin adaptor proteins implicated in clathrin-mediated protein sorting between the trans-Golgi network and endosomes. GGA RNAi knockdowns in Drosophila have resulted in conflicting data concerning whether the Drosophila GGA (dGGA) is essential. The goal of this study was to define the null phenotype for the unique Drosophila GGA. We describe two independently derived dGGA mutations. Neither allele expresses detectable dGGA protein. Homozygous and hemizygous flies with each allele are viable and fertile. In contrast to a previous report using RNAi knockdown, GGA mutant flies show no evidence of age-dependent retinal degeneration or cathepsin missorting. Our results demonstrate that several of the previous RNAi knockdown phenotypes were the result of off-target effects. However, GGA null flies are hypersensitive to dietary chloroquine and to starvation, implicating GGA in lysosomal function and autophagy.
References
[1]
Hirst J, Robinson MS (1998) Clathrin and adaptors. Biochim Biophys Acta 1404: 173–193.
[2]
Robinson MS (2004) Adaptable adaptors for coated vesicles. Trends Cell Biol 14: 167–174.
[3]
Boman AL, Zhang C, Zhu X, Kahn RA (2000) A family of ADP-ribosylation factor effectors that can alter membrane transport through the trans-Golgi. Mol Biol Cell 11: 1241–1255.
[4]
Poussu A, Lohi O, Lehto VP (2000) Vear, a novel Golgi-associated protein with VHS and gamma-adaptin "ear" domains. J Biol Chem 275: 7176–7183.
[5]
Dell'Angelica EC, Puertollano R, Mullins C, Aguilar RC, Vargas JD, et al. (2000) GGAs: a family of ADP ribosylation factor-binding proteins related to adaptors and associated with the Golgi complex. J Cell Biol 149: 81–94.
[6]
Hirst J, Lui WW, Bright NA, Totty N, Seaman MN, et al. (2000) A family of proteins with gamma-adaptin and VHS domains that facilitate trafficking between the trans-Golgi network and the vacuole/lysosome. J Cell Biol 149: 67–80.
[7]
Takatsu H, Yoshino K, Nakayama K (2000) Adaptor gamma ear homology domain conserved in gamma-adaptin and GGA proteins that interact with gamma-synergin. Biochem Biophys Res Commun 271: 719–725.
[8]
Takatsu H, Katoh Y, Shiba Y, Nakayama K (2001) Golgi-localizing, gamma-adaptin ear homology domain, ADP-ribosylation factor-binding (GGA) proteins interact with acidic dileucine sequences within the cytoplasmic domains of sorting receptors through their Vps27p/Hrs/STAM (VHS) domains. J Biol Chem 276: 28541–28545.
[9]
Puertollano R, Aguilar RC, Gorshkova I, Crouch RJ, Bonifacino JS (2001) Sorting of mannose 6-phosphate receptors mediated by the GGAs. Science 292: 1712–1716.
[10]
Puertollano R, Bonifacino JS (2004) Interactions of GGA3 with the ubiquitin sorting machinery. Nat Cell Biol 6: 244–251.
[11]
Shiba Y, Katoh Y, Shiba T, Yoshino K, Takatsu H, et al. (2004) GAT (GGA and Tom1) domain responsible for ubiquitin binding and ubiquitination. J Biol Chem 279: 7105–7111.
[12]
Bilodeau PS, Winistorfer SC, Allaman MM, Surendhran K, Kearney WR, et al. (2004) The GAT domains of clathrin-associated GGA proteins have two ubiquitin binding motifs. J Biol Chem 279: 54808–54816.
[13]
Bonifacino JS (2004) The GGA proteins: adaptors on the move. Nat Rev Mol Cell Biol 5: 23–32.
[14]
Wasiak S, Legendre-Guillemin V, Puertollano R, Blondeau F, Girard M, et al. (2002) Enthoprotin: a novel clathrin-associated protein identified through subcellular proteomics. J Cell Biol 158: 855–862.
[15]
Mattera R, Arighi CN, Lodge R, Zerial M, Bonifacino JS (2003) Divalent interaction of the GGAs with the Rabaptin-5-Rabex-5 complex. EMBO J 22: 78–88.
[16]
Lui WW, Collins BM, Hirst J, Motley A, Millar C, et al. (2003) Binding partners for the COOH-terminal appendage domains of the GGAs and gamma-adaptin. Mol Biol Cell 14: 2385–2398.
[17]
Zhu Y, Doray B, Poussu A, Lehto VP, Kornfeld S (2001) Binding of GGA2 to the lysosomal enzyme sorting motif of the mannose 6-phosphate receptor. Science 292: 1716–1718.
[18]
Puertollano R, Randazzo PA, Presley JF, Hartnell LM, Bonifacino JS (2001) The GGAs promote ARF-dependent recruitment of clathrin to the TGN. Cell 105: 93–102.
[19]
Costaguta G, Stefan CJ, Bensen ES, Emr SD, Payne GS (2001) Yeast Gga coat proteins function with clathrin in Golgi to endosome transport. Mol Biol Cell 12: 1885–1896.
[20]
Hirst J, Lindsay MR, Robinson MS (2001) GGAs: roles of the different domains and comparison with AP-1 and clathrin. Mol Biol Cell 12: 3573–3588.
[21]
Kamikura DM, Cooper JA (2006) Clathrin interaction and subcellular localization of Ce-DAB-1, an adaptor for protein secretion in Caenorhabditis elegans. Traffic 7: 324–336.
[22]
Mardones GA, Burgos PV, Brooks DA, Parkinson-Lawrence E, Mattera R, et al. (2007) The trans-Golgi network accessory protein p56 promotes long-range movement of GGA/clathrin-containing transport carriers and lysosomal enzyme sorting. Mol Biol Cell 18: 3486–3501.
[23]
Hida T, Ikeda H, Kametaka S, Akazawa C, Kohsaka S, et al. (2007) Specific depletion of GGA2 causes cathepsin D missorting in HeLa cells. Arch Histol Cytol 70: 303–312.
[24]
Ghosh P, Griffith J, Geuze HJ, Kornfeld S (2003) Mammalian GGAs act together to sort mannose 6-phosphate receptors. J Cell Biol 163: 755–766.
[25]
Hirst J, Sahlender DA, Choma M, Sinka R, Harbour ME, et al. (2009) Spatial and Functional Relationship of GGAs and AP-1 inDrosophilaand HeLa Cells. Traffic 10: 1696–1710.
[26]
He X, Li F, Chang WP, Tang J (2005) GGA proteins mediate the recycling pathway of memapsin 2 (BACE). J Biol Chem 280: 11696–11703.
[27]
Govero J, Doray B, Bai H, Kornfeld S (2012) Analysis of Gga null mice demonstrates a non-redundant role for mammalian GGA2 during development. PLoS One 7: e30184.
[28]
Ghosh P, Kornfeld S (2003) Phosphorylation-induced conformational changes regulate GGAs 1 and 3 function at the trans-Golgi network. J Biol Chem 278: 14543–14549.
[29]
Dennes A, Cromme C, Suresh K, Kumar NS, Eble JA, et al. (2005) The novel Drosophila lysosomal enzyme receptor protein mediates lysosomal sorting in mammalian cells and binds mammalian and Drosophila GGA adaptors. J Biol Chem 280: 12849–12857.
[30]
Kametaka S, Sawada N, Bonifacino JS, Waguri S (2010) Functional characterization of protein-sorting machineries at the trans-Golgi network in Drosophila melanogaster. J Cell Sci 123: 460–471.
[31]
Kametaka S, Kametaka A, Yonekura S, Haruta M, Takenoshita S, et al. (2012) AP-1 clathrin adaptor and CG8538/Aftiphilin are involved in Notch signaling during eye development in Drosophila melanogaster. J Cell Sci 125: 634–648.
[32]
Eissenberg JC, Ilvarsonn AM, Sly WS, Waheed A, Krzyzanek V, et al. (2011) Drosophila GGA model: an ultimate gateway to GGA analysis. Traffic 12: 1821–1838.
[33]
Hirst J, Carmichael J (2011) A potential role for the clathrin adaptor GGA in Drosophila spermatogenesis. BMC Cell Biol 12: 22.
Chen H, Ma Z, Liu Z, Tian Y, Xiang Y, et al. (2009) Case studies of ends-out gene targeting in Drosophila. Genesis 47: 305–308.
[36]
Schug J, Schuller WP, Kappen C, Salbaum JM, Bucan M, et al. (2005) Promoter features related to tissue specificity as measured by Shannon entropy. Genome Biol 6: R33.
[37]
Myllykangas L, Tyynela J, Page-McCaw A, Rubin GM, Haltia MJ, et al. (2005) Cathepsin D-deficient Drosophila recapitulate the key features of neuronal ceroid lipofuscinoses. Neurobiol Dis 19: 194–199.
[38]
Guarnieri FG, Arterburn LM, Penno MB, Cha Y, August JT (1993) The motif Tyr-X-X-hydrophobic residue mediates lysosomal membrane targeting of lysosome-associated membrane protein 1. J Biol Chem 268: 1941–1946.
[39]
Pulipparacharuvil S, Akbar MA, Ray S, Sevrioukov EA, Haberman AS, et al. (2005) Drosophila Vps16A is required for trafficking to lysosomes and biogenesis of pigment granules. J Cell Sci 118: 3663–3673.
[40]
Kurz DJ, Decary S, Hong Y, Erusalimsky JD (2000) Senescence-associated (beta)-galactosidase reflects an increase in lysosomal mass during replicative ageing of human endothelial cells. J Cell Sci 113 (Pt 20): 3613–3622.
[41]
Seglen PO, Grinde B, Solheim AE (1979) Inhibition of the lysosomal pathway of protein degradation in isolated rat hepatocytes by ammonia, methylamine, chloroquine and leupeptin. Eur J Biochem 95: 215–225.
[42]
Simonsen A, Birkeland HC, Gillooly DJ, Mizushima N, Kuma A, et al. (2004) Alfy, a novel FYVE-domain-containing protein associated with protein granules and autophagic membranes. J Cell Sci 117: 4239–4251.
[43]
Finley KD, Edeen PT, Cumming RC, Mardahl-Dumesnil MD, Taylor BJ, et al. (2003) blue cheese mutations define a novel, conserved gene involved in progressive neural degeneration. J Neurosci 23: 1254–1264.
[44]
Simonsen A, Cumming RC, Lindmo K, Galaviz V, Cheng S, et al. (2007) Genetic modifiers of the Drosophila blue cheese gene link defects in lysosomal transport with decreased life span and altered ubiquitinated-protein profiles. Genetics 176: 1283–1297.
[45]
Lim A, Kraut R (2009) The Drosophila BEACH family protein, blue cheese, links lysosomal axon transport with motor neuron degeneration. J Neurosci 29: 951–963.
[46]
Mizushima N (2007) Autophagy: process and function. Genes Dev 21: 2861–2873.
[47]
Scott RC, Schuldiner O, Neufeld TP (2004) Role and regulation of starvation-induced autophagy in the Drosophila fat body. Dev Cell 7: 167–178.
[48]
Kramer JM, Slade JD, Staveley BE (2008) foxo is required for resistance to amino acid starvation in Drosophila. Genome 51: 668–672.
[49]
Simonsen A, Cumming RC, Finley KD (2007) Linking lysosomal trafficking defects with changes in aging and stress response in Drosophila. Autophagy 3: 499–501.
[50]
Mizushima N, Levine B (2010) Autophagy in mammalian development and differentiation. Nat Cell Biol 12: 823–830.
[51]
Tsukamoto S, Kuma A, Murakami M, Kishi C, Yamamoto A, et al. (2008) Autophagy is essential for preimplantation development of mouse embryos. Science 321: 117–120.
[52]
Kuma A, Hatano M, Matsui M, Yamamoto A, Nakaya H, et al. (2004) The role of autophagy during the early neonatal starvation period. Nature 432: 1032–1036.
[53]
Gelfman CM, Vogel P, Issa TM, Turner CA, Lee WS, et al. (2007) Mice lacking alpha/beta subunits of GlcNAc-1-phosphotransferase exhibit growth retardation, retinal degeneration, and secretory cell lesions. Invest Ophthalmol Vis Sci 48: 5221–5228.
