Intracellular trafficking and secretion of adiponectin is dependent on GGA-coated vesicles.
Adiponectin (Acrp30) is an insulin-sensitizing hormone produced and secreted exclusively by adipose tissue. Confocal fluorescent microscopy demonstrated the colocalization of adiponectin with the Golgi membrane markers p115, beta-COP, and the trans-Golgi network marker, syntaxin 6. Treatment of cells with brefeldin A redistributed adiponectin to the endoplasmic reticulum where it colocalized with the chaperone protein BIP and inhibited secretion of adiponectin demonstrating a requirement for a functional Golgi apparatus for adiponectin release. Confocal fluorescent microscopy also demonstrated a colocalization of endogenous adiponectin with that of expressed GGA1myc (Golgi-localizing gamma-adaptin ear homology ARF-binding protein) but with no significant overlap between adiponectin and the GGA2myc or GGA3myc isoforms. Consistent with confocal fluorescent microscopy, transmission electron microscopy demonstrated the colocalization of GGA1 with adiponectin. Although GGA1 did not directly interact with the adiponectin protein, the adiponectin enriched membrane compartments of adipocyte were precipitated by a GST-GGA1 cargo binding domain (VHS) fusion protein but not with a GST-GGA2 VHS or GST-GGA3 VHS fusion proteins. Moreover, co-expression of adiponectin with a GGA1 dominant-interfering mutant (GGA1-VHS GAT domain) resulted in a marked inhibition of adiponectin secretion in both 3T3L1 adipocytes and HEK293 cells, whereas no inhibition was detected with the truncated mutants GGA2-VHSGAT or GGA3-VHSGAT. Moreover, co-expression of wild type GGA1 with adiponectin enhanced secretion of adiponectin. Interestingly, leptin secretion was unaffected by neither the wild type form or GGA1 mutant. Taken together these data demonstrate that the trafficking of adiponectin through its secretory pathway is dependent on GGA-coated vesicles.
Xie
Linglin
L
Division of Biology, Kansas State University, Manhattan, KS 66506, USA.
Boyle
Daniel
D
Sanford
Daniel
D
Scherer
Philipp E
PE
Pessin
Jeffrey E
JE
Mora
Silvia
S
D041721:3T3-L1 Cells; D020727:ADP-Ribosylation Factors / Q000378:metabolism / Q000502:physiology*; D033942:Adaptor Proteins, Vesicular Transport / Q000378:metabolism / Q000502:physiology*; D017667:Adipocytes / Q000378:metabolism; D052242:Adiponectin / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport; D020126:Brefeldin A / Q000737:chemistry; D002460:Cell Line; D003513:Cycloheximide / Q000494:pharmacology; D004247:DNA / Q000737:chemistry; D018274:Electroporation; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005799:Genes, Dominant; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007328:Insulin / Q000378:metabolism; D020738:Leptin / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism; D051379:Mice; D018613:Microscopy, Confocal; D046529:Microscopy, Electron, Transmission; D008856:Microscopy, Fluorescence; D009154:Mutation; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D021381:Protein Transport; D050765:Qa-SNARE Proteins / Q000378:metabolism; D011863:Radioimmunoassay; D011994:Recombinant Proteins / Q000378:metabolism; D013696:Temperature; D013997:Time Factors; D014162:Transfection; D014759:Viral Envelope Proteins / Q000378:metabolism; D021601:trans-Golgi Network
D033942:Adaptor Proteins, Vesicular Transport; D052242:Adiponectin; C028391:G protein, vesicular stomatitis virus; C407306:GGA adaptor proteins; D007328:Insulin; D020738:Leptin; D008562:Membrane Glycoproteins; D020033:Protein Isoforms; D011500:Protein Synthesis Inhibitors; D050765:Qa-SNARE Proteins; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; D049452:Green Fluorescent Proteins; D020126:Brefeldin A; D004247:DNA; D003513:Cycloheximide; D005982:Glutathione Transferase; D020727:ADP-Ribosylation Factors