GGA function is required for maturation of neuroendocrine secretory granules.
Secretory granule (SG) maturation has been proposed to involve formation of clathrin-coated vesicles (CCVs) from immature SGs (ISGs). We tested the effect of inhibiting CCV budding by using the clathrin adaptor GGA (Golgi-associated, gamma-ear-containing, ADP-ribosylation factor-binding protein) on SG maturation in neuroendocrine cells. Overexpression of a truncated, GFP-tagged GGA, VHS (Vps27, Hrs, Stam)-GAT (GGA and target of myb (TOM))-GFP led to retention of MPR, VAMP4, and syntaxin 6 in mature SGs (MSGs), suggesting that CCV budding from ISGs is inhibited by the SG-localizing VHS-GAT-GFP. Furthermore, VHS-GAT-GFP-overexpression disrupts prohormone convertase 2 (PC2) autocatalytic cleavage, processing of secretogranin II to its product p18, and the correlation between PC2 and p18 levels. All these effects were not observed if full-length GGA1-GFP was overexpressed. Neither GGA1-GFP nor VHS-GAT-GFP perturbed SG protein budding from the TGN, or homotypic fusion of ISGs. Reducing GGA3 levels by using short interfering (si)RNA also led to VAMP4 retention in SGs, and inhibition of PC2 activity. Our results suggest that inhibition of CCV budding from ISGs downregulates the sorting from the ISGs and perturbs the intragranular activity of PC2.
Kakhlon
Or
O
Secretory Pathway Laboratories, Cancer Research UK, London Research Institute, London, UK.
Sakya
Prabhat
P
Larijani
Banafshe
B
Watson
Rose
R
Tooze
Sharon A
SA
D020727:ADP-Ribosylation Factors / Q000235:genetics / Q000378:metabolism*; D033942:Adaptor Proteins, Vesicular Transport / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002864:Chromogranins / Q000378:metabolism; D022163:Clathrin-Coated Vesicles / Q000378:metabolism / Q000502:physiology*; D015536:Down-Regulation; D049452:Green Fluorescent Proteins / Q000235:genetics; D008561:Membrane Fusion; D009154:Mutation; D016716:PC12 Cells; D045706:Proprotein Convertase 2 / Q000378:metabolism; D021381:Protein Transport; D050765:Qa-SNARE Proteins / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D017527:Receptor, IGF Type 2 / Q000378:metabolism; D022142:Secretory Vesicles / Q000378:metabolism / Q000502:physiology*
D033942:Adaptor Proteins, Vesicular Transport; D002864:Chromogranins; C407306:GGA adaptor proteins; D050765:Qa-SNARE Proteins; D034741:RNA, Small Interfering; D017527:Receptor, IGF Type 2; D049452:Green Fluorescent Proteins; D045706:Proprotein Convertase 2; D020727:ADP-Ribosylation Factors