The interactome of the UapA transporter reveals putative new players in anterograde membrane cargo trafficking

Georgiou, Xenia; Dimou, Sofia; Diallinas, George; Samiotaki, Martina

doi:10.1016/j.fgb.2023.103840

Cited by 5 publications

References 85 publications

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A type II phosphatidylinositol-4-kinase coordinates sorting of cargo polarizing by endocytic recycling

Kourkoulou,

Martzoukou,

Fischer

et al. 2024

Commun Biol

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Depending on their phosphorylation status, derivatives of phosphatidylinositol play important roles in vesicle identity, recognition and intracellular trafficking processes. In eukaryotic cells, phosphatidylinositol-4 phosphate pools generated by specific kinases are key determinants of the conventional secretion pathways. Earlier work in yeast has classified phosphatidylinositol-4 kinases in two types, Stt4p and Pik1p belonging to type III and Lsb6p to type II, with distinct cellular localizations and functions. Eurotiomycetes appear to lack Pik1p homologues. In Aspergillus nidulans, unlike homologues in other fungi, AnLsb6 is associated to late Golgi membranes and when heterologously overexpressed, it compensates for the thermosensitive phenotype in a Saccharomyces cerevisiae pik1 mutant, whereas its depletion leads to disorganization of Golgi-associated PHOSBP-labelled membranes, that tend to aggregate dependent on functional Rab5 GTPases. Evidence provided herein, indicates that the single type II phosphatidylinositol-4 kinase AnLsb6 is the main contributor for decorating secretory vesicles with relevant phosphatidylinositol-phosphate species, which navigate essential cargoes following the route of apical polarization via endocytic recycling.

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A type II phosphatidylinositol-4-kinase coordinates sorting of cargo polarizing by endocytic recycling

Kourkoulou,

Martzoukou,

Fischer

et al. 2024

Commun Biol

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Neosynthesized polarized and non-polarized plasma membrane cargoes follow distinct trafficking routes inAspergillus nidulans

Sagia,

Georgiou,

Chamilos

et al. 2024

Preprint

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Membrane proteins are thought to be sorted to the plasma membrane (PM) via Golgi-dependent trafficking. However, our recent studies in the fungusAspergillus nidulanschallenged the essentiality of Golgi in the biogenesis of non-polarly localized transporters and receptors. Here, we investigate the mechanism of trafficking of membrane proteins, by following the localization of a polar cargo (R-SNARE SynA) versus a non-polar cargo (UapA transporter), synchronously co-expressed in wild-type or isogenic genetic backgrounds repressible for conventional cargo secretion. In wild-type, the two cargoes dynamically label distinct secretory compartments, highlighted by the observation that, unlike SynA, UapA does not colocalize with the late-Golgi. In line with partitioning into distinct early secretory carriers, UapA and SynA translocation to the PM is differentially dependent on Sec13, and importantly the two cargoes collapse in distinct early secretory compartments in a sec31ts mutant or upon CopA repression. Trafficking via distinct cargo-specific carriers is further supported by the observation that repression or inactivation of key proteins essential for late-Golgi /TGN maturation and post-Golgi vesicular secretion did not affect proper trafficking of UapA, but totally blocked SynA secretion. Surprisingly, several specific SNARE proteins that are absolutely essential for conventional cargo vesicular secretion, as well as the exocyst effector RabDSec4, proved dispensable for UapA translocation to the PM. Our findings point to a model where UapA proper trafficking and insertion into the PM might involve non-canonical SNARE combinations. Overall, the present work establishes unequivocally the existence of distinct, cargo-dependent, trafficking mechanisms, initiating at early secretory compartments.

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Locus-specific chromatin proteomics using dCas-guided proximity labelling inAspergillus nidulans

Svoboda,

Niederdöckl-Loibl,

Schüller

et al. 2024

Preprint

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Proximity labelling that uses promiscuous biotin ligases (BirA) fused to a bait protein is a powerful tool to identify protein interaction partners in vivo under different metabolic or developmental conditions. BirA can also be used to determine protein composition and interaction partners at specific chromatin locations when it is fused with enzymatically-disabled Cas9 (dCas9) and then guided to the location of interest by sgRNAs. We adapted this method (called CasID) for fungal cells using the nitrate assimilation gene cluster of A. nidulans as a model locus and estrogen-inducible expression of the dCas9-BirA fusion to improve condition-specific labelling. For method establishment, we first verified the presence of dCas-BirA and a known transcription factor at the nitrate locus by chromatin immunoprecipitation (ChIP). Results show that both dCas-BirA and the AreA transcription factor are present at the locus of interest under the conditions used for biotinylation. We then optimized the CasID procedure for efficient labelling and background reduction using the CasID-sgRNA strain and two control strains, one lacking the sgRNA and another one lacking the whole CasID system. Here we provide proof-of-concept for the suitability of the method by showing that biotinylated proteins are enriched in the CasID strains in comparison to the controls. After background reduction, 32 proteins remained in two independent experiments exclusively enriched in the Cas-ID-sgRNA strain. Among these proteins was NmrA, an AreA-interacting regulator, and we also found several chromatin-associated proteins. Overall, our results demonstrate that Cas-ID is suitable for locus-specific labelling and identification of chromatin-associated proteins and transcription factors in A. nidulans. However, the high background of proteins that are biotinylated out of chromatin context or unspecifically attach to the affinity purification matrix needs to be addressed by implementing a set of rigorous controls. In summary, we herewith provide a detailed protocol for application of the method that proved to be useful for the identification of novel chromatin-associated proteins and their interaction partners at a specific genomic locus in divers metabolic and developmental conditions.

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The interactome of the UapA transporter reveals putative new players in anterograde membrane cargo trafficking

Cited by 5 publications

References 85 publications

A type II phosphatidylinositol-4-kinase coordinates sorting of cargo polarizing by endocytic recycling

A type II phosphatidylinositol-4-kinase coordinates sorting of cargo polarizing by endocytic recycling

Neosynthesized polarized and non-polarized plasma membrane cargoes follow distinct trafficking routes inAspergillus nidulans

Locus-specific chromatin proteomics using dCas-guided proximity labelling inAspergillus nidulans

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