Tracking exocytosis of a <scp>GPI</scp>‐anchored protein in <i>Aspergillus nidulans</i>

Peñalva, Miguel; Moscoso-Romero, Esteban; Hernández‐González, Miguel

doi:10.1111/tra.12761

Cited by 10 publications

(7 citation statements)

References 48 publications

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“…Overall, our previous ( Dimou et al, 2020 ) and present results show that several nutrient transporters, the major H + pump ATPase PmaA and a component of a pH sensor, all use a similar Golgi-independent route to translocate from the ER to the PM. This route is clearly distinct from the well-established Golgi- and microtubule-dependent trafficking of cargoes needed for A. nidulans polar growth, including chitin synthase ChsB ( Fukuda et al, 2009 ; Hernández-González et al, 2018a ), the synaptobrevin-like secretory v-SNARE SynA ( Taheri-Talesh et al, 2008 ; Pantazopoulou and Peñalva, 2011 ; Martzoukou et al, 2018 ), the lipid flippases DnfA and DnfB ( Schultzhaus et al, 2015 , 2017 ; Martzoukou et al, 2018 ), the glycosylphosphatidylinositol-anchored protein (GPI-AP) EglC ( Peñalva et al, 2020 ), or the soluble extracellular inulinase InuA ( Hernández-González et al, 2018b ). The membrane cargoes SynA, ChsB, DnfA, and DnfB all show strict polar localization at the Spitzenkörper/SPK [a vesicle supply apical center; ( Zhou et al, 2018 )] and the apical plasma membrane, co-localize dynamically with Golgi markers, and their biogenesis is aberrant in conditional mutants of key Golgi proteins (e.g., SedV, HypA, HypB, Tlg2, or RabO).…”

Section: Discussionmentioning

confidence: 97%

Golgi-Bypass Is a Major Unconventional Route for Translocation to the Plasma Membrane of Non-Apical Membrane Cargoes in Aspergillus nidulans

Dimou

Dionysopoulou

Sagia

et al. 2022

Front. Cell Dev. Biol.

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Nutrient transporters have been shown to translocate to the plasma membrane (PM) of the filamentous fungus Aspergillus nidulans via an unconventional trafficking route that bypasses the Golgi. This finding strongly suggests the existence of distinct COPII vesicle subpopulations, one following Golgi-dependent conventional secretion and the other directed towards the PM. Here, we address whether Golgi-bypass concerns cargoes other than nutrient transporters and whether Golgi-bypass is related to cargo structure, size, abundance, physiological function, or polar vs. non-polar distribution in the PM. To address these questions, we followed the dynamic subcellular localization of two selected membrane cargoes differing in several of the aforementioned aspects. These are the proton-pump ATPase PmaA and the PalI pH signaling component. Our results show that neosynthesized PmaA and PalI are translocated to the PM via Golgi-bypass, similar to nutrient transporters. In addition, we showed that the COPII-dependent exit of PmaA from the ER requires the alternative COPII coat subunit LstA, rather than Sec24, whereas PalI requires the ER cargo adaptor Erv14. These findings strengthen the evidence of distinct cargo-specific COPII subpopulations and extend the concept of Golgi-independent biogenesis to essential transmembrane proteins, other than nutrient transporters. Overall, our findings point to the idea that Golgi-bypass might not constitute a fungal-specific peculiarity, but rather a novel major and cargo-specific sorting route in eukaryotic cells that has been largely ignored.

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Section: Discussionmentioning

confidence: 97%

Golgi-Bypass Is a Major Unconventional Route for Translocation to the Plasma Membrane of Non-Apical Membrane Cargoes in Aspergillus nidulans

Dimou

Dionysopoulou

Sagia

et al. 2022

Front. Cell Dev. Biol.

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“…Traffic blocks resulting from RAB impairment can be used to discriminate the exocytic pathways followed by different cargoes. For example, secretion of both the soluble enzyme inulinase and the GPI‐anchored protein EglC is dependent on hypA1 affecting Trs120 in TRAPPII (implying that is dependent on RAB11), but inulinase absolutely requires RAB5 whereas GPI‐EglC does not (Hernández‐González et al., 2018b; Peñalva et al., 2020), indicating that the latter travels directly from the Golgi to the PM, whereas the former undergoes some type of diversion through EEs before being delivered to the extracellular milieu. The chitin synthase ChsB recycles through endosomes, but its traffic is completely unaffected by RAB5 ablation, indicating that it travels to the Golgi through a primary sorting endosome located functionally upstream of the RAB5 domain (Hernández‐González et al., 2018a) (see Figure 2).…”

Section: The “Canonical” Post‐golgi Rabs: Rab11 and Sec4mentioning

confidence: 99%

The fungal RABOME: RAB GTPases acting in the endocytic and exocytic pathways of Aspergillus nidulans (with excursions to other filamentous fungi)

Pinar

Peñalva

2021

Molecular Microbiology

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RABs (ras-related in brain) comprise a family of small GTPases belonging, with RAS, RHO, RAN, and ARF families to the RAS superfamily (Rojas et al., 2012). RABs were originally described in Saccharomyces cerevisiae, with Ypt1 (the homolog of metazoan RAB1), formerly denoted YPT (for "yeast protein two"), holding the honor of being the first RAB ever identified, and the founding member of the clan (Gallwitz et al., 1983;Schmitt et al., 1986;Segev & Botstein, 1987). Involvement of the yeast exocytic RABs Ypt1 and Sec4 in intracellular traffic was discovered shortly afterwards (Salminen & Novick, 1987;Segev et al., 1988). Meanwhile, genes encoding proteins closely related to yeast YPTs were identified in a rat cDNA library, showing that these GTPases are ubiquitous in eukaryotes (Touchot et al., 1987). In the filamentous fungal world RAB GTPases have been systematically investigated in Aspergillus nidulans, which will be used as guiding thread for this review, with relevant contributions from Ustilago maydis (where the longdistance movement of RAB5 early endosomes (EEs) was originally reported), Magnaporthe oryzae (where Sec4 and its effector, the exocyst, are involved in pathogenesis), and Neurospora crassa (which is well suited to study the spatial regulation of exocytic RABs in the Spitzenkörper), discussed when appropriate.The jungle of gene nomenclature characterizing many fungal papers has also permeated the RAB field. Regretfully, this nomenclature problem jeopardizes literature searches, often impacting

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“…We reasoned that increasing expression would result in E6K/G540S GHD supporting growth over a wider range of temperatures. Thus, we drove its expression with the promoter of the inulinase inuA gene, which is inducible by the presence of sucrose in the medium and almost completely shut off on glucose (Hernández-González et al, 2018; Peñalva et al, 2020). Initially we tested wild-type and E6K/G540S GHD in a uso1+ background.…”

Section: Resultsmentioning

confidence: 99%

Tethering by Uso1 is dispensable: The Uso1 monomeric globular head domain interacts with SNAREs to maintain viability

Bravo-Plaza

Tagua

Arst

et al. 2022

Preprint

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Uso1/p115 and RAB1 tether ER-derived vesicles to the Golgi. Uso1/p115 contains a globular-head-domain (GHD), a coiled-coil (CC) mediating dimerization/tethering and a C-terminal region (CTR) interacting with golgins. Uso1/p115 is recruited to vesicles by RAB1. Paradoxically, genetic studies placed Uso1 acting upstream of, or in conjunction with RAB1 (Sapperstein et al., 1996). We selected two missense mutations in uso1 resulting in E6K and G540S substitutions in the GHD permitting growth of otherwise inviable rab1-deficient Aspergillus nidulans. Remarkably, the double mutant suppresses the complete absence of RAB1. Full-length Uso1 and CTRΔ proteins are dimeric and the GHD lacking the CC/CTR is monomeric irrespective of whether they carry or not E6K/G540S. Microscopy showed recurrence of Uso1 on puncta (60 sec half-life) colocalizing with RAB1 and less so with early Golgi markers Sed5 and GeaA/Gea1/Gea2. Localization of Uso1 but not of Uso1E6K/G540S to puncta is abolished by compromising RAB1 function, indicating that E6K/G540S creates interactions bypassing RAB1. By S-tag-coprecipitation we demonstrate that Uso1 is an associate of the Sed5/Bos1/Bet1/Sec22 SNARE complex zippering vesicles with the Golgi, with Uso1E6K/G540S showing stronger association. Bos1 and Bet1 bind the Uso1 GHD directly, but Bet1 is a strong E6K/G540S-independent binder, whereas Bos1 is weaker but becomes as strong as Bet1 when the GHD carries E6K/G540S. AlphaFold2 predicts that G540S actually increases binding of GHD to the Bos1 Habc domain. In contrast, E6K seemingly increases membrane targeting of an N-terminal amphipathic a-helix, explaining phenotypic additivity. Overexpression of E6K/G540S and wild-type GHD complemented uso1Δ. Thus, a GHD monomer provides the essential Uso1 functions, demonstrating that long-range tethering activity is dispensable. Therefore, when enhanced by E6K/G540S, Uso1 binding to Bos1/Bet1 required to regulate SNAREs bypasses both the contribution of RAB1 to Uso1 recruitment and the reported role of RAB1 in SNARE complex formation (Lupashin and Waters, 1997), suggesting that the latter is consequence of the former.

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Tracking exocytosis of a GPI‐anchored protein in Aspergillus nidulans

Cited by 10 publications

References 48 publications

Golgi-Bypass Is a Major Unconventional Route for Translocation to the Plasma Membrane of Non-Apical Membrane Cargoes in Aspergillus nidulans

Golgi-Bypass Is a Major Unconventional Route for Translocation to the Plasma Membrane of Non-Apical Membrane Cargoes in Aspergillus nidulans

The fungal RABOME: RAB GTPases acting in the endocytic and exocytic pathways of Aspergillus nidulans (with excursions to other filamentous fungi)

Tethering by Uso1 is dispensable: The Uso1 monomeric globular head domain interacts with SNAREs to maintain viability

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