COPI localizes to the early Golgi in Aspergillus nidulans

Abstract

Coatomer-I (COPI) is a heteromeric protein coat that facilitates the budding of membranous carriers mediating Golgi-to-ER and intra-Golgi transport. While the structural features of COPI have been thoroughly investigated, its physiological role is insufficiently understood. Here we exploit the amenability of A. nidulans for studying intracellular traffic, taking up previous studies by Breakspear et al. (2007) with the -COP/CopA subunit of COPI. Endogenously tagged -COP/CopA largely localizes to SedV Sed5 syntaxin-containing early Golgi cisterna, and acute inactivation of ER-to-Golgi traffic delocalizes COPI to a haze, consistent with the cisternal maturation model. In contrast, the Golgi localization of COPI is independent of the TGN regulators HypB Sec7 and HypA Trs120 , implying that COPI budding predominates at the SedV Sed5 early Golgi, with lesser contribution of the TGN. This finding agrees with the proposed role of COPI-mediated intra-Golgi retrograde traffic in driving cisternal maturation, which predicts that the capacity of the TGN to generate COPI carriers is low. The COPI early Golgi compartments intimately associates with Sec13-containing ER exit sites. Characterization of the heat-sensitive copA1ts (sod VI C1) mutation showed that it results in a single residue substitution in the -COP-binding Carboxyl-Terminal-Domain of -COP that likely destabilizes its folding. However, we show that Golgi disorganization by copA1ts necessitates > 150 min-long incubation at 42ºC. This weak subcellular phenotype makes it unsuitable for inactivating COPI traffic acutely for microscopy studies, and explains the aneuploidy-stabilizing role of the mutation at subrestrictive temperatures.

Functional exploration of co-expression networks identifies a nexus for modulating protein and citric acid titres in Aspergillus niger submerged culture

“…Accordingly, we retrieved candidates which are co-expressed with genes involved in the TCA cycle (citrate synthase citA , fumarate reductase fumR , and isocitrate dehydrogenase idh2 ), and vesicle trafficking at the Golgi (alpha/beta subunits of the coat protein complex (COPI) copA / sec26 , and COPII subunit sec13 ). The COPI and COPII complex mediate retrograde and anterograde vesicle trafficking between the Golgi and endoplasmic reticulum, respectively [26, 27]. Note that all six query genes were also selected due to evidence of function based on wet-lab experimentation conducted in either A. niger or A. nidulans [28].…”

Characterization of Aspergillus nidulans TRAPPs uncovers unprecedented similarities between fungi and metazoans and reveals the modular assembly of TRAPPII

“…We modified a previous method [54,56]. Conidiospore suspensions of strains expressing Bet5-S, Trs120-S, Trs65-S, Trs85-S or Tca17-S from endogenously tagged alleles were inoculated in MFA (Aspergillus fermentation medium) consisting of SC supplemented with 2.5% (v/ v) of corn steep liquor syrup (Solulys 048R, Roquette Laisa S.A., Valencia, Spain), 50 mM each of Na 2 HPO 4 and NaH 2 PO 4 , 50 mM NaCl, 3% (w/v) sucrose as main C source, 20 mM (NH 4 ) 2 SO 4 as main N source and vitamins as required to supplement the auxotrophic mutations present in each strain.…”

Tracking exocytosis of a GPI‐anchored protein in Aspergillus nidulans

“…S. cerevisiae GPI‐APs accumulate in discrete ERES containing the Sec24 homolog Lst1 upon blocking COPII biogenesis with an outer COPII coat sec31‐1 ts mutation 5,8 . In A. nidulans ERES appear as numerous punctate structures dispersed across the cytosol and polarized toward the apex, 46 a pattern that was not observed in micrographs of EglC::GFP::GPI expressed in hyphae in which COPII vesicle biogenesis had been blocked with sarA6 (Figure 3A, Supp. Figure S3 and Movie 4).…”