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.

Life and Death of Fungal Transporters under the Challenge of Polarity

“…In particular, we followed transporter localization in strains that did not express Sec24 or Sec13 (COPII generation), SedV Sed5 or GeaA Gea1 (early-Golgi functioning), HypB Sec7 (TGN functioning), RabE Rab11 , AP-1 σ or clathrin ClaH Chc1 (post-Golgi secretion), RabA/B Rab5 (early and recycling endosomes), or SsoA Sso1 (major PM tethering t-SNARE). Notice that although persistent transcriptional repression (i.e., >24 h) of secretion eventually leads to A. nidulans cell death, given that in our system full repression needed 10-12 h to occur, this provided a period of time for conidiospore germination and the development of germlings, and also sufficient time, after full repression of secretion, for inducing and studying the trafficking of transporters [20]. Besides using strains where proteins of the secretory route could be repressed, we also examined transporter traffic in the presence of drugs that block microtubule or actin filament polymerization (benomyl and Latrunculin B, respectively), processes reported as essential in conventional secretion.…”

“…Still, other transporters do not define PM microdomains (e.g., the general amino acid permease Gap1, several sugar transporters, etc.). In A. nidulans, several nucleobase transporters of the NCS1 family also do not define microdomains, at least within the limits of conventional epifluorescence microscopy [59][60][61][62], but members of the AzgA and NAT/NCS2 family appear as distinct foci when expressed at moderate levels [20,25,63]. In general, however, there is no evidence that transporters in fungi localize in a polar fashion.…”

“…In some cases, internalized transporters are also detected in highly motile transient cytoplasmic structures, which proved to be early endosomes [70,71]. Some transporters can also be detected marking the perinuclear or cortical ER, but this occurs only when overexpressed via strong promoters [20]. Partially misfolded transporters due to mutations are also blocked in the ER [25].…”

“…For both approaches, we primarily used, as a model transporter cargo, the well-studied uric acid-xanthine UapA transporter. Subsequently, we also examined the trafficking of other nutrient transporters, namely AzgA (purines) and FurA (allantoin), representing structurally and functionally distinct transporter families [20].…”

“…If we consider that clathrin coats transporter vesicles at some point, this suggests there must be some 'missing' events of uncoating (COPII) and coating (clathrin), before the fusion of vesicles to the PM. This made us consider the possibility of formation of an intermediate compartment between the ERes and the PM, which is probably too transient for detection with standard fluorescence microcopy [20]. Interestingly, clathrin light chain has been found to be redundant for the forward trafficking of both transporters and apical cargoes [18], but it is essential specifically for the endocytosis of transporters ( [17]; see later).…”