Dissection of GTPase-activating proteins reveals functional asymmetry in the COPI coat of budding yeast

Abstract

The Arf GTPase controls formation of the COPI vesicle coat. Recent structural models of COPI revealed the positioning of two Arf1 molecules in contrasting molecular environments. Each of these pockets for Arf1 is expected to also accommodate an Arf GTPase-activating protein (ArfGAP). Structural evidence and protein interactions observed between isolated domains indirectly suggest that each niche preferentially recruits one of the two ArfGAPs known to affect COPI, i.e. Gcs1/ArfGAP1 and Glo3/ArfGAP2/3, although only partial structures are available. The functional role of the unique non-catalytic domain of either ArfGAP has not been integrated into the current COPI structural model. Here, we delineate key differences in the consequences of triggering GTP hydrolysis through the activity of one versus the other ArfGAP. We demonstrate that Glo3/ArfGAP2/3 specifically triggers Arf1 GTP hydrolysis impinging on the stability of the COPI coat. We show that the Snf1 kinase complex, the yeast homologue of AMP-activated protein kinase (AMPK), phosphorylates the region of Glo3 that is crucial for this effect and, thereby, regulates its function in the COPI-vesicle cycle. Our results revise the model of ArfGAP function in the molecular context of COPI.

AP‐3 vesicle uncoating occurs after HOPS‐dependent vacuole tethering

“…Arf GAPs may play a critical role here. They function in cargo concentration at budding sites of vesicles as shown for Sar1 (Futai et al, 2004;Tabata et al, 2009) and Gcs1 (Arakel et al, 2019), and promote uncoating, likely in coordination with the tethering and fusion machinery, as suggested for Age2 here. How the coat and uncoating machinery as well as fusion machinery cooperate and regulate each other's activity is an exciting and rather unexpected avenue to be explored in the trafficking field.…”

“…In this regard, the interaction of the ER-localized Dsl1 tethering complex with the COPI coat (Andag & Schmitt, 2003;Ren et al, 2009;Meiringer et al, 2011) is intriguing as it suggests a crosstalk of a coated vesicle with a tether . Recent work on COPI revealed a close interplay of two ArfGAPs, Glo3, and Gcs1 in uncoating, one of which is controlled in activity by phosphorylation (Arakel et al, 2019). COPI binds two Arf1 proteins in different molecular environments (Bykov et al, 2017;Dodonova et al, 2017), as does AP-1 (Shen et al, 2015), suggesting that one Arf GAP protein may destabilize the coat partially.…”

“…The increase in the number of AP-3 vesicles in the age2 deletion led us to postulate that Age2 is required for proper uncoating of the vesicles, as this was shown for other ArfGAPs before (Arakel et al, 2019). We thus overexpressed Age2 or Gcs1 using a TEF-promoter to test for possible premature uncoating and thus a defect in AP-3 trafficking using the GNS construct.…”

“…Age2 is a potential uncoating factor in the AP-3 pathway GAP proteins conduct different functions in adaptor-mediated vesicle formation, ranging from cargo enrichment in COPII trafficking (Tabata et al, 2009) to vesicle uncoating in COPI trafficking via Gcs1 and Glo3 (Arakel et al, 2019). Age2 is one of four GAPs of Arf1 present at the Golgi and functions at the TGN (Fig 6A) (Poon et al, 2001).…”

The p24 Complex Contributes to Specify Arf1 for COPI Coat Selection

“…First, p24 proteins could initially recruit the deactivated form of Arf1 (Arf1-GDP) [ 23 ], facilitating its interaction with the pre-assembled Gea1/COPI complex to be subsequently activated and directed for COPI binding. Furthermore, the direct interaction of p24 proteins with the ArfGAP Glo3 [ 27 ], which is stably associated to COPI, could contribute to regulate its functions in COPI vesicle biogenesis and uncoating [ 34 , 35 , 36 ]. Therefore, we suggest that the ability of p24 proteins to oligomerize could contribute to establish multiple, transient interactions with the different regulatory elements of the COPI vesicle machinery, favoring their local encounter on the cis -Golgi membrane for productive vesicle budding ( Figure 7 ).…”