GBF/Gea mutant with a single substitution sustains fungal growth in the absence of BIG/Sec7

Abstract

HighlightsA. nidulans has a GBF/Gea and a BIG/Sec7 subfamily Golgi Arf1-GEFs, both essential.The late Golgi Arf1-GEF mutant hypB5 conditionally blocks secretion.Residue substitution in the early Golgi Arf1-GEF GeaA suppresses hypB5 and hypBΔ.The mutation alters a GBF/Gea amino acid motif and shifts GeaA localization.GeaA1 alone satisfies the eukaryotic requirement for two Golgi Arf1 GEFs.

The Golgi apparatus: insights from filamentous fungi

“…BFA has predictably two targets at the A. nidulans Golgi: the two Golgi Arf1 GEFs, that is GeaA, homolog of the early Golgi GBF1/Gea1p and HypB Sec7, homolog of the late Golgi BIG/Sec7p. However, although shifting the conditional late Golgi hypB5 sec7ts mutant (Yang et al 2008, Arst et al 2014) to the restrictive temperature resulted in GFP-RabE relocalizing from the SPZ to abnormal membrane aggregations in the proximity of the nucleus, it did not mimic the dispersion to the cytoplasm elicited by BFA. This suggests that RabE recruitment to membranes depends on a determinant acting upstream of (or independent of) HypB Sec7 and downstream of the early BFA target, the Golgi Arf1-GEF GeaA.…”

“…2A). GeaA:: GFP, a member of the family of early Golgi Arf1-GEFs (GBF1/Gea), also co-localized substantially with mCherry::SedV, while segregating from mRFP::PH OSBP (Arst et al 2014). GFP::TlgB (TlgB is the Tlg2/ syntaxin16 homolog) co-localized with mRFP::PH OSBP at the late territory, while mRFP::TlgB segregated from GFP::SedV (Pinar et al 2013).…”

“…2B). The markers we used were functionally suggestive: The only two A. nidulans Golgi syntaxins SedV and Tlg2 (López-Berges et al 2016) mark two distinct compartments by the conserved Arf1-GEFs GeaA and Sec7, members of the cis/early and trans/ late subfamily of Golgi Arf1-GEFs, respectively, (Arst et al 2014) co-localize with the relevant early or late syntaxin; the RerA receptor responsible for the retrieval of ER resident proteins early in the secretory pathway (Sato et al 1997) is confined with the early Golgi syntaxin and the early Golgi Arf1-GEF, as expected; the coincidence of PI4P with activated Arf1, both of which have a pivotal role in Golgi exit (D'SouzaSchorey and Chavrier 2006, Daboussi et al 2012, De Matteis et al 2013, is detected by the PH OSBP reporter only where the late Golgi syntaxin and the late Arf1-GEF reside. It is important to note that for each of the compartments both transmembrane (SedV, RerA, Tlg2) and peripheral membrane proteins (GeaA, Sec7, PH OSBP ) were tagged and localized, minimizing the possibility of artifacts related to the membraneassociation mechanism of the hybrid proteins.…”

“…Both Arf1-GEFs in A. nidulans, HypB Sec7 and GeaA, are virtually essential (Arst et al 2014). Using genetics once again, extragenic suppressors of the conditional late Golgi Arf1 GEF mutant hypB5 were sought (Arst et al 2014).…”

“…Using genetics once again, extragenic suppressors of the conditional late Golgi Arf1 GEF mutant hypB5 were sought (Arst et al 2014). Among lots of intragenic suppressors, we recovered a single extragenic one that carries a mutation in the early Golgi Arf1 GEF, GeaA.…”