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Translocation of nutrient transporters to cell membrane via Golgi bypass in Aspergillus nidulans.

ABSTRACT: Nutrient transporters, being polytopic membrane proteins, are believed, but not formally shown, to traffic from their site of synthesis, the ER, to the plasma membrane through Golgi-dependent vesicular trafficking. Here, we develop a novel genetic system to investigate the trafficking of a neosynthesized model transporter, the well-studied UapA purine transporter of Aspergillus nidulans. We show that sorting of neosynthesized UapA to the plasma membrane (PM) bypasses the Golgi and does not necessitate key Rab GTPases, AP adaptors, microtubules or endosomes. UapA PM localization is found to be dependent on functional COPII vesicles, actin polymerization, clathrin heavy chain and the PM t-SNARE SsoA. Actin polymerization proved to primarily affect COPII vesicle formation, whereas the essential role of ClaH seems indirect and less clear. We provide evidence that other evolutionary and functionally distinct transporters of A. nidulans also follow the herein identified Golgi-independent trafficking route of UapA. Importantly, our findings suggest that specific membrane cargoes drive the formation of distinct COPII subpopulations that bypass the Golgi to be sorted non-polarly to the PM, and thus serving house-keeping cell functions.

SUBMITTER: Dimou S

PROVIDER: S-EPMC7332984 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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Translocation of nutrient transporters to cell membrane via Golgi bypass in Aspergillus nidulans.

Dimou Sofia S Martzoukou Olga O Dionysopoulou Mariangela M Bouris Vangelis V Amillis Sotiris S Diallinas George G

EMBO reports 20200526 7

Nutrient transporters, being polytopic membrane proteins, are believed, but not formally shown, to traffic from their site of synthesis, the ER, to the plasma membrane through Golgi-dependent vesicular trafficking. Here, we develop a novel genetic system to investigate the trafficking of a neosynthesized model transporter, the well-studied UapA purine transporter of Aspergillus nidulans. We show that sorting of neosynthesized UapA to the plasma membrane (PM) bypasses the Golgi and does not neces ...[more]

PMID: 32452614

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Project description:We exploited the amenability of the fungus Aspergillus nidulans to genetics and live-cell microscopy to investigate autophagy. Upon nitrogen starvation, GFP-Atg8-containing pre-autophagosomal puncta give rise to cup-shaped phagophores and circular (0.9-μm diameter) autophagosomes that disappear in the vicinity of the vacuoles after their shape becomes irregular and their GFP-Atg8 fluorescence decays. This 'autophagosome cycle' gives rise to characteristic cone-shaped traces in kymographs. Autophagy does not require endosome maturation or ESCRTs, as autophagosomes fuse with vacuoles directly in a RabS (homolog of Saccharomyces cerevisiae Ypt7 and mammalian RAB7; written hereafter as RabS(RAB7))-HOPS-(homotypic fusion and vacuole protein sorting complex)-dependent manner. However, by removing RabS(RAB7) or Vps41 (a component of the HOPS complex), we show that autophagosomes may still fuse, albeit inefficiently, with the endovacuolar system in a process almost certainly mediated by RabA(RAB5)/RabB(RAB5) (yeast Vps21 homologs)-CORVET (class C core vacuole/endosome tethering complex), because acute inactivation of HbrA/Vps33, a key component of HOPS and CORVET, completely precludes access of GFP-Atg8 to vacuoles without affecting autophagosome biogenesis. Using a FYVE 2-GFP probe and endosomal PtdIns3P-depleted cells, we imaged PtdIns3P on autophagic membranes. PtdIns3P present on autophagosomes decays at late stages of the cycle, preceding fusion with the vacuole. Autophagy does not require Golgi traffic, but it is crucially dependent on RabO(RAB1). TRAPPIII-specific factor AN7311 (yeast Trs85) localizes to the phagophore assembly site (PAS) and RabO(RAB1) localizes to phagophores and autophagosomes. The Golgi and autophagy roles of RabO(RAB1) are dissociable by mutation: rabO(A136D) hyphae show relatively normal secretion at 28°C but are completely blocked in autophagy. This finding and the lack of Golgi traffic involvement pointed to the ER as one potential source of membranes for autophagy. In agreement, autophagosomes form in close association with ring-shaped omegasome-like ER structures resembling those described in mammalian cells.

Dataset Information

Translocation of nutrient transporters to cell membrane via Golgi bypass in Aspergillus nidulans.

Publications

Translocation of nutrient transporters to cell membrane via Golgi bypass in Aspergillus nidulans.

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