Abstract
SummaryAlthough adaptor protein complex 1 (AP-1) and Golgi-localized, γ ear-containing, ADP-ribosylation factor-binding proteins (GGAs) are both adaptors for clathrin-mediated intracellular trafficking, the pathways they mediate and their relationship to each other remain open questions [1]. To tease apart the functions of AP-1 and GGAs, we rapidly inactivated each adaptor using the “knocksideways” system [2] and then compared the protein composition of clathrin-coated vesicle (CCV) fractions from control and knocksideways cells. The AP-1 knocksideways resulted in a dramatic and unexpected loss of GGA2 from CCVs. Over 30 other peripheral membrane proteins and over 30 transmembrane proteins were also depleted, including several mutated in genetic disorders, indicating that AP-1 acts as a linchpin for intracellular CCV formation. In contrast, the GGA2 knocksideways affected only lysosomal hydrolases and their receptors. We propose that there are at least two populations of intracellular CCVs: one containing both GGAs and AP-1 for anterograde trafficking and another containing AP-1 for retrograde trafficking. Our study shows that knocksideways and proteomics are a powerful combination for investigating protein function, which can potentially be used on many different types of proteins.
Highlights
Rerouting of adaptor protein complex 1 (AP-1) and GGA Adaptors to Mitochondria The clathrin adaptor AP-1 is expressed in all eukaryotes and has been implicated in pathways as diverse as sorting of lysosomal hydrolases and/or their receptors in mammals [3, 4], protein trafficking to olfactory cilia in C. elegans [5], biogenesis of rhoptry organelles in Toxoplasma [6], formation of contractile vacuoles in Dictyostelium [7], and protein localization to the trans-Golgi network (TGN) in yeast [8]
Our results indicate that AP-1 acts as a linchpin in the formation of intracellular clathrincoated vesicle (CCV), and that if AP-1 is acutely lost, these vesicles cannot form
One of the CCV components that was most strongly dependent on AP-1 was GGA2. This result challenges the conclusions of a recent study carried out on yeast, which proposed that GGAs and AP-1 nucleate the formation of different populations of CCVs [11], but it is consistent with immunoelectron microscopy studies showing that at least some clathrin-coated-budding profiles are positive for both AP-1 and GGAs [9, 10]
Summary
Addition of rapamycin for 10 min caused g-FKBP to be strongly depleted from CCVs, similar to endogenous g in a conventional AP-1 knockdown (Figures 1B and 1C, left). All three of these proteins were substantially depleted from CCVs isolated from AP-1 knocksideways cells, and we saw a partial loss of clathrin, as previously reported [2] (Figure 1C; see Figure S1 available online).
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