Functional characterization of protein-sorting machineries at the trans-Golgi network in Drosophila melanogaster

Abstract

SummaryTargeting of proteins to their final destination is a prerequisite for living cells to maintain their homeostasis. Clathrin functions as a coat that forms transport carriers called clathrin-coated vesicles (CCVs) at the plasma membrane and post-Golgi compartments. In this study, we established an experimental system using Schneider S2 cells derived from the fruit fly, Drosophila melanogaster, as a model system to study the physiological roles of clathrin adaptors, and to dissect the processes of CCV formation. We found that a clathrin adaptor Drosophila GGA (dGGA), a homolog of mammalian GGA proteins, localizes to the trans-Golgi network (TGN) and is capable of recruiting clathrin from the cytosol onto TGN membranes. dGGA itself is recruited from the cytosol to the TGN in an ARF1 small GTPase (dARF79F)-dependent manner. dGGA recognizes the cytoplasmic acidic-cluster-dileucine (ACLL) sorting signal of Lerp (lysosomal enzyme receptor protein), a homolog of mammalian mannose 6-phosphate receptors. Moreover, both dGGA and another type of TGN-localized clathrin adaptor, AP-1 (adaptor protein-1 complex), are shown to be involved in the trafficking of Lerp from the TGN to endosomes and/or lysosomes. Taken together, our findings indicate that the protein-sorting machinery in fly cells is well conserved relative to that in mammals, enabling the use of fly cells to dissect CCV biogenesis and clathrin-dependent protein trafficking at the TGN of higher eukaryotes.

Drosophila rugoseIs a Functional Homolog of MammalianNeurobeachinand Affects Synaptic Architecture, Brain Morphology, and Associative Learning

“…Anti-Rg staining also displayed a granular pattern reminiscent of the Golgi network (Fig. 1 I), a cellular structure that is dispersed throughout the cell in fruit flies (Kametaka et al, 2010). Using the cis-Golgi marker GM130, we demonstrated that Drosophila Rg resides near or within the Golgi complex (Fig.…”

Type II phosphatidylinositol 4-kinase regulates trafficking of secretory granule proteins inDrosophila

“…Indeed, partial loss-of-function AP-1 mutants exhibit small granules similar to those found in PI4KII mutants (Burgess et al, 2011). Alternatively, PI4KII might affect the Golgi recruitment of other PI4P-binding proteins involved in post-Golgi vesicular trafficking, for example GGA or GOLPH3 (Dippold et al, 2009;Hirst et al, 2009;Kametaka et al, 2010). Nonetheless, the overwhelming majority of Sgs3 appears to traffic normally to small secretory granules, indicating that post-Golgi trafficking of glue cargo proteins is relatively unaffected in PI4KII mutants.…”

Regulation of β2-Adrenergic Receptor Maturation and Anterograde Trafficking by an Interaction with Rab Geranylgeranyltransferase

“…Mutating the L 339 L 340 motif in the ␤ 2 AR did not change ligand binding, G protein coupling, or signaling to adenylate cyclase (36), indicating that decreased anterograde trafficking of the F(X) 6 AA mutant receptor was not caused by improper receptor folding. Clathrin-associated proteins such as the heterotetrameric adaptor protein (AP) complexes and the monomeric Golgi-localized, ␥-adaptin ear-containing, ARF-binding proteins (GGAs) function as "adaptors," promoting both concentration of cargo and recruitment of clathrin at sites of transport-carrier formation (40). AP-1 and GGAs, in particular, localize to the TGN and endosomes and participate in protein trafficking between these two compartments (40 -42).…”