Translocation of nutrient transporters to cell membrane via Golgi bypass in Aspergillus nidulans
2020; Springer Nature; Volume: 21; Issue: 7 Linguagem: Inglês
10.15252/embr.201949929
ISSN1469-3178
AutoresSofia Dimou, Όλγα Μαρτζούκου, Mariangela Dionysopoulou, Vangelis Bouris, Sotiris Amillis, George Diallinas,
Tópico(s)Microtubule and mitosis dynamics
ResumoArticle26 May 2020free access Transparent processSource Data Translocation of nutrient transporters to cell membrane via Golgi bypass in Aspergillus nidulans Sofia Dimou Department of Biology, National and Kapodistrian University of Athens, Athens, Greece Search for more papers by this author Olga Martzoukou orcid.org/0000-0001-5445-484X Department of Biology, National and Kapodistrian University of Athens, Athens, Greece Search for more papers by this author Mariangela Dionysopoulou Department of Biology, National and Kapodistrian University of Athens, Athens, Greece Search for more papers by this author Vangelis Bouris Department of Biology, National and Kapodistrian University of Athens, Athens, Greece Search for more papers by this author Sotiris Amillis Department of Biology, National and Kapodistrian University of Athens, Athens, Greece Search for more papers by this author George Diallinas Corresponding Author [email protected] orcid.org/0000-0002-3426-726X Department of Biology, National and Kapodistrian University of Athens, Athens, Greece Search for more papers by this author Sofia Dimou Department of Biology, National and Kapodistrian University of Athens, Athens, Greece Search for more papers by this author Olga Martzoukou orcid.org/0000-0001-5445-484X Department of Biology, National and Kapodistrian University of Athens, Athens, Greece Search for more papers by this author Mariangela Dionysopoulou Department of Biology, National and Kapodistrian University of Athens, Athens, Greece Search for more papers by this author Vangelis Bouris Department of Biology, National and Kapodistrian University of Athens, Athens, Greece Search for more papers by this author Sotiris Amillis Department of Biology, National and Kapodistrian University of Athens, Athens, Greece Search for more papers by this author George Diallinas Corresponding Author [email protected] orcid.org/0000-0002-3426-726X Department of Biology, National and Kapodistrian University of Athens, Athens, Greece Search for more papers by this author Author Information Sofia Dimou1, Olga Martzoukou1, Mariangela Dionysopoulou1, Vangelis Bouris1, Sotiris Amillis1 and George Diallinas *,1 1Department of Biology, National and Kapodistrian University of Athens, Athens, Greece *Corresponding author. Tel: +30 210 7274649; E-mail: [email protected] EMBO Rep (2020)21:e49929https://doi.org/10.15252/embr.201949929 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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. Synopsis Nutrient transporter translocation to the cell membrane operates via a novel trafficking route that does not involve functioning of the Golgi in fungi. Transporter translocation from the endoplasmic reticulum (ER) to the plasma membrane (PM) depends on COPII vesicle formation. Golgi and known post-Golgi secretory routes are non-essential for transporter biogenesis. The mechanisms regulating trafficking of nutrient transporters and apical cargoes are distinct. Introduction Plasma membrane (PM) transporters mediating the selective cellular uptake or efflux of solutes and drugs are essential proteins in all organisms. The first step in their biogenesis, being polytopic transmembrane proteins, is their co-translational translocation into the membrane of the endoplasmic reticulum (ER). The current belief is that after translocation into the ER, transporters are sorted into nascent ER-exit sites, pack into budding COPII vesicles that fuse to the cis-Golgi and then reach the trans-Golgi network (TGN) via Golgi maturation 1-3. From the TGN, transporters are thought to be secreted towards the PM, similar to other membrane cargoes, either directly or indirectly via the endosomal compartment, in AP-1/clathrin coated vesicles, the trafficking of which is controlled by multiple Rab GTPases and the microtubule cytoskeleton 4, 5. However, some lines of evidence support that specific transporters might not follow known conventional Golgi and post-Golgi dependent routes. For example, genetic knock-out of proteins involved in TGN-dependent membrane cargo sorting (e.g. Arfrp1, golgin-160 or AP-1) leads to accumulation of the insulin-regulated GLUT4 glucose carrier in the PM, rather than retention in the Golgi or other intracellular compartments, suggesting the presence of alternative routes out of the TGN, or even Golgi-independent mechanisms 6. In addition, kinesin motor proteins or microtubule disruption has a moderate or no effect on GLUT4 accumulation at the PM 7, 8. Additionally, it has been shown recently that neosynthesized GLUT4 is sorted to the PM from an early secretory compartment, bypassing the TGN 9. Noticeably also, a specific form of the CFTR transmembrane protein (ΔF508-CFTR), an ATP-binding cassette (ABC) transporter that functions as a low conductance Cl− selective channel associated with cystic fibrosis, has been formally shown to translocate to the PM via Golgi bypass under specific stress conditions 10, 11. Finally, the mammalian potassium channel Kv2.1 is known to be sorted to the PM of the initial segment (AIS) of neurons via a mechanism that bypasses the Golgi 12. In fact, no formal evidence exists on whether neosynthesized transporters traffic through the Golgi/TGN compartment in any type of cell. A possible explanation for this might be that transporter passage and exit from the Golgi is very rapid, never leading to accumulation of sufficient steady-state levels for detection with standard fluorescence microscopy. However, evidence against this explanation is also the fact that no mutation or specific condition has been shown to block PM transporters in the Golgi. In contrast, several mutations affecting the proper folding or altering specific motifs in transporters are well-known to lead to retention in the ER, which is often associated with ubiquitination-dependent turnover by proteasome degradation and/or selective autophagy 13, 14. Unconventional trafficking routes that bypass the Golgi have also been described for a handful of PM transmembrane proteins other than transporters and are collectively classified as type IV unconventional protein secretion (UPS) 10, 11, 15-17. In the course of experiments addressing cargo trafficking in the model fungus Aspergillus nidulans, we noticed that sorting to the PM of the well-studied UapA transporter 18 is not affected by repression of transcription of the AP-1 adaptor complex, a key effector of conventional secretion of most membrane cargoes 19. This result contrasted to those obtained with polarly localized membrane cargoes (e.g. Chitin synthase ChsB, Synaptobrevin SynA, or lipid flippases DfnA or DfnB), which all needed AP-1 for their apical localization 20, 21. Interestingly, endocytosis of UapA and other nutrient transporters has been shown to involve a mechanism distinct from that of apical membrane cargoes. In particular, transporters are internalized all along the hyphal membrane via a clathrin-dependent, but AP-2-independent mechanism, destined for vacuolar degradation in response to physiological or stress signals 19. In contrast, polar cargoes are constitutively recycled in the apical region of hyphae via clathrin-independent, but AP-2-dependent, endocytosis, a process essential for polarity maintenance and filamentous polar growth 19, 20. Thus, current evidence suggests that subcellular trafficking routes of nutrient transporters, which are localized homogenously along the hyphal membrane, might be mechanistically distinct from that of polarly localized apical membrane cargoes. Several additional observations prompted us to dissect the mechanism of biogenesis of transporters. First, we have never obtained any genetic or microscopic evidence that A. nidulans transporters pass from Golgi-like structures. Second, all A. nidulans transporters studied in our lab (more then 30, all relative to uptake of nutrients such as purines, pyrimidines, amino acids and carboxylic acids) are not glycosylated. A similar observation holds true for most nutrient transporters studied in Saccharomyces cerevisiae. Finally, in cases where specific transporters in other systems have been reported to localize in the trans-Golgi network (TGN), the experiments described do not distinguish whether this is the result of transporter recycling from the PM or secretion of de novo made molecules from the ER. In the present work, we used UapA as a model cargo to investigate its dynamic trafficking to the PM. For doing so, we developed a controllable genetic system to study the trafficking of de novo made UapA when different steps of the conventional secretion pathway are tightly repressed. This system, combined with relative co-localization studies of UapA and molecular markers of the secretory pathway, showed that UapA localization to the PM occurs without the need of Golgi- or post-Golgi-dependent cargo sorting mechanisms. We further showed that UapA localization to the PM is dependent on the formation of COPII vesicles, clathrin, actin polymerization and the PM t-SNARE SsoA. To extend our findings, we also performed key experiments with additional transporters of A. nidulans and found that in all cases, their trafficking mechanism is similar to that identified with UapA. We finally discuss our findings within the general context of trafficking of nutrient transporter in polarized eukaryotic cells. Results In vivo trafficking of neosynthesized UapA We established conditions to follow ab initio the traffic and subcellular localization of de novo made transporters in single hyphae of A. nidulans. As a prototype transporter-cargo, we used the well-studied UapA purine transporter, functionally tagged with GFP 18. Conidiospores of an otherwise wild-type A. nidulans strain, containing an in-locus targeted uapA-gfp allele, were allowed to germinate overnight (14–16 h, 25°C) under conditions that repress uapA-gfp transcription. This is achieved using either the native uapA promoter or the regulatable alcAp promoter (for details, see Materials and Methods). After this period, uapA-gfp transcription was induced in germlings (i.e. very young hyphae) via a shift to derepressing conditions (0–8 h). Derepressed protein levels of UapA-GFP driven by either the native or the alcAp promoter are very similar, at all-time points. The strategy for repression–derepression of UapA synthesis is depicted in Fig 1A. Figure 1. Subcellular localization of neosynthesized UapA A. Cartoon depicting the strategy for following the trafficking of neosynthesized UapA. The subcellular localization of UapA is shown in green. Red circles indicate positioning of nuclei. (for more details, see main text). B. In vivo epifluorescence microscopy following de novo expressed UapA-GFP in a single growing germling at 70, 80, 100, 120, 140, 160 and 170 min after derepression of transcription via its native uapA promoter (for details, see text and Materials and Methods). Notice that in the growing germling tip UapA localizes in a membranous mesh and some cytosolic puncta, whereas in more tip-distal parts of the germlings, UapA is in PM-localized puncta (see white arrows), which progressively become more abundant so that at more posterior areas the PM is homogeneously labelled. C. In vivo epifluorescence microscopy following de novo expressed UapA-GFP in a germling maturing to young hypha at 180, 240, 300, 360 and 400 min after derepression of transcription via its native uapA promoter. During this developmental transition, UapA apical localization is gradually diminished. As the cell tip acquires a faster growth rate, UapA is completely retracted from the hyphal apex (see zoom-in panels on the right and white arrows). D. In a mature hyphal cell, UapA is no longer found in the PM of the extreme apical compartment, but in a cytoplasmic membrane network resembling the ER. As the distance from the apex increases, UapA progressively populates the PM as cortical puncta (see white arrows). E. Cartoon depicting the rearrangement of actin and UapA at the tip of A. nidulans during germling to hyphal maturation. In germlings, dense arrays of actin cables are present in the apex, known as apical actin array (AAA). Actin patches are concentrated, but not restricted, to the apex. In mature hyphae, a core of actin appears in the position of the Spitzenkorper and actin patches are restricted to the subapical endocytic collar (≈2 μm behind the apex). Actin cables are retracted from the apex and shifted to a more distal region of the hyphal tip, the subapical actin web (SAW). In the apex, actin cables are now found associated mostly as sparse arrays across the cell cortex. The localization of UapA during hyphal maturation seems to follow the pattern of actin cables 26, 27. F. In vivo epifluorescence microscopy following neosynthesized alcAp-UapA-GFP in a single hypha at 120, 130, 140, 150, 160 and 170 min, under overexpressing (derepression/ethanol-induction) conditions, as described in Materials and Methods. Notice that UapA labels the perinuclear ER rings (white arrows). G. In vivo epifluorescence microscopy following de novo expressed alcAp-GFP-SynA in a single young hyphal cell, at 60, 70, 80, 90 and 100 min after transcriptional derepression (for strain details, see Materials and Methods). SynA is a standard polar membrane cargo that traffics to the hyphal tip via Golgi-dependent secretion. Notice the very distinct GFP fluorescent signals obtained using UapA (non-polar membrane network and PM) versus SynA (Golgi-like puncta and polar depositioning at the tip; see also alter). H. Similar experiment as in (B) performed in an isogenic strain where ArtA, the arrestin required for UapA endocytosis, is genetically depleted. Data information: For (B, C, D, F, G and H), the picture shown represents similar results obtained by following several hyphae. Scale bars: 5 μm. Source data are available online for this figure. Source Data for Figure 1 [embr201949929-sup-0003-SDataFig1.zip] Download figure Download PowerPoint Figure 1B and C shows examples of images obtained following UapA-GFP localization, when expressed from its native promoter, in single germlings (young hyphae). Similar results were obtained in several experiments. UapA-GFP appears as cytoplasmic weak fluorescence at 70 min after the onset of transcription. Subsequently, UapA-GFP labels a membranous cytoplasmic network (80–120 min) resembling A. nidulans ER membranes 14, 22 and progressively migrates to the cell periphery, where it appears as dispersed cortical puncta (120–160 min). With time, cortical puncta increase in number to eventually label the entire PM of hyphae in a rather homogeneous manner (170 min). The timing of appearance of UapA molecules in the periphery of cells is compatible with previous studies which show that UapA transport activity reaches its steady state 3 h after transcriptional derepression 23. Importantly, we did not detect fluorescence-labelled cytoplasmic structures resembling early or late Golgi compartments (see later). Relatively increased accumulation of UapA in the PM of the growing apical region (or in the apex of secondary germ tubes emerging from the conidiospore head) was observed in young germlings (see 140- to 170-min samples in Fig 1B). Noticeably, as germlings grow longer, the relatively increased apical cortical localization of UapA is progressively diminished (Fig 1C, see 240–400 min), and in longer more mature hyphae is lost (Fig 1D). Thus, in long hyphae, UapA is imaged to clearly label an ER-like membranous network at the apical region, but in subapical regions, it progressively populates the PM at distinct puncta that are rather unevenly distributed. Interestingly, the change in the localization of UapA at the apical region is co-incident to important cellular changes that underlie the developmental transition from slow-growing germlings to fast-growing mature hyphae observed in A. nidulans and other filamentous ascomycetes 24. Transition to 5- to 10-fold faster apical growth is related to the formation of an apical secretory vesicular organizing centre, known as the Spitzenkörper, and re-organization of apical actin cables from a dense network (in germlings) to less dense and cortical localization (in mature hyphae) (25, 26; also depicted in Fig 1E). During this transition, an apical actin array also retracts to a subapical actin web, compatible with the change in the apical localization of UapA in germlings versus mature hyphae, respectively 26, 27. These observations will become more apparent later, when actin polymerization is shown to be essential for UapA trafficking. Overall, results highlighted in Fig 1B–D suggested that neosynthesized UapA labels the ER, as probably expected, but it then appears in the PM with no indication of passing from other recognizable cytoplasmic structures, such as the Golgi or motile endosomes 28. To acquire additional evidence that UapA labels mostly the ER and not Golgi-like structures, we also followed the localization of de novo made UapA-GFP under conditions that the transporter is significantly overexpressed. For this, we made use of the alcAp promoter expressed under conditions of derepression plus ethanol-induction (for details, see Materials and Methods). Results shown in Fig 1F revealed that UapA overexpression labels, in addition to a diffuse membrane network, the characteristic perinuclear ER rings that are often seen in fungi (see 130- to 170-min samples). Notably, however, UapA-GFP overexpression did not label Golgi-like or other punctuate structures. In addition to UapA, we also examined the dynamic localization of a standard de novo made apical marker, such as Synaptobrevin A tagged with GFP (GFP-SynA; 25). In this case, unlike UapA, we were able to detect SynA in several Golgi-like and other punctuate cytoplasmic structures (Fig 1G). Some of the punctuate structures at later time points, when SynA has already reached its apical localization, showed endosome-like motility, as expected due to apical recycling 25. Thus, the picture of UapA versus SynA secretion was strikingly different, the former marking mostly the ER network and eventually the PM, the latter marking the ER, Golgi-like or other punctuate structures, and the tip of the apical region. Notably, UapA appearance in the PM does not seem to occur via lateral diffusion from the tip area. This is concluded based on two observations. First, there is no apparent continuous gradient of UapA from the apical area towards subapical parts, but instead isolated UapA cortical puncta (see arrows in Fig 1B, at 140 and 160 min). Second, the tip region in mature hyphae possesses practically no UapA, while the subapical compartments show several cortical and unevenly distributed UapA puncta (Fig 1D). These observations are also compatible with previous reports showing that membrane later diffusion of large transmembrane proteins is extremely slow or short distance 29, 30. Thus, the simplest scenario is that UapA localization to the PM takes place by short-range lateral sorting from the ER network. Previous studies have shown that UapA can be endocytosed and degraded in the vacuole in response to its activity or due physiological signals 31, 32. The latter study has also strongly supported that internalized UapA is not recycled back to the PM after endocytosis. Also, later in this work, we further show that UapA localization is independent of the function of recycling endosomes (see Appendix Fig S3). Thus, the subcellular localization of UapA shown in Fig 1B–D reflects strictly secretion of neosynthesized UapA. To exclude any doubt on the role of endocytosis and recycling in the images obtained, we repeated here the microscopic analysis in a strain that is genetically blocked in UapA ubiquitination and endocytosis, and thus to possible recycling, due to a null mutation in the specific arrestin adaptor ArtA 32. The result, shown in Fig 1H, was practically identical to the one obtained in the wild-type strain. Neosynthesized UapA does not co-localize with Golgi markers To obtain stronger evidence that de novo made UapA does not pass from the Golgi compartments as suggested by results highlighted in Fig 1, we performed co-localization studies of UapA-GFP with Golgi-specific molecular markers tagged with mRFP or mCherry. In A. nidulans, fluorescent-tagged protein markers distinguishing “early” (corresponding to cis) and “late” (corresponding to trans) Golgi have been very rigorously established 21, 33. Early Golgi is commonly marked with SedVSed5 or GeaAGea1 and late Golgi/TGN with HypBSec7 or PHOSBP. Both compartments appear as numerous, rather immotile, cytoplasmic puncta, all along the length of hyphae (see cartoon in lower left panel of Fig 2). Both compartments are transient, consistent with the cisternae maturation model, where late Golgi has an average lifetime of approximately 2-3 min 34. Figure 2A and B shows representative time course experiments of trafficking of neosynthesized UapA-GFP, driven by the alcAp promoter, in strains co-expressing either SedV (early Golgi) or PHOSBP (late Golgi/TGN). In both cases, we did not detect significant co-localization of UapA with the Golgi markers used. The result depicted in Fig 2A and B was statistically supported as Pearson correlation coefficients for co-localization were very low in both cases (PCC < 0.35, P < 0.0001). Notice that Pearson correlation coefficients ≤ 0.35 are often obtained in co-localization studies of markers of distinct cytoplasmic compartments that are topologically close (e.g. ER and Golgi). Thus, our findings are taken as a significant indication that UapA might not pass from the Golgi. Figure 2. Neosynthesized UapA does not co-localize with Golgi markers A, B. Co-localization analysis of neosynthesized alcAp-UapA-GFP with early (SedV) or late (PHOSBP) markers, respectively, tagged with mCherry or mRFP. Images show single hyphal cells. For conditions of transcriptional derepression, see Materials and Methods. Quantification of co-localization was performed by calculating Pearson's correlation coefficient (PCC). One sample t-test was performed to test the significance of differences in PCCs. Biological/technical replicates:2/9 for alcAp-UapA-GFP mCherry-SedV and 2/8 for alcAp-UapA-GFP mRFP-PHOSBP. For the definition of the two categories of replicates, see Materials and Methods. Results of quantification, shown on the middle, suggest that there is no significant overlapping fluorescent signal of UapA with SedV (PCC = 0.25 ± 0.09, P < 0.0001) or with PHOSBP (PCC = 0.34 ± 0.06, P < 0.0001), as PCC values close to 0.2–0.3 are also commonly obtained when distinct compartments (e.g. ER and early Golgi or early and late Golgi) are followed with different fluorophores. Scale bars: 2 μm. C, D. Co-localization analysis of neosynthesized alcAp-GFP-SynA, used as a conventional cargo that traffics through the Golgi, with early (SedV) or late (PHOSBP) markers tagged with mCherry or mRFP, respectively. Images show single hyphal cells. For conditions of transcriptional derepression, see Materials and Methods. Quantification of co-localization and statistical analysis was performed as in (A, B). Biological/technical replicates: 2/10 for each strain. Statistical analysis showed significant co-localization of SynA with the late Golgi marker (PCC = 0.67 ± 0.04, P < 0.0001), but not with early Golgi marker (PCC = 0.37 ± 0.06, P < 0.0001). An explanation for lack of co-localization of SynA with SedV is given in the text. Scale bars: 2 μm. The cartoon at the bottom depicts the localization of early or late markers as established here in several previous studies (see 20, 21). Source data are available online for this figure. Source Data for Figure 2 [embr201949929-sup-0004-SDataFig2.zip] Download figure Download PowerPoint To obtain more concrete evidence that we have not missed the time window for passage of UapA from the Golgi, we repeated essentially the same co-localization experiment using a standard secreted cargo, namely SynA (synaptobrevin A), which is localized polarly in the apical region of hyphae via the conventional Golgi-dependent pathway 21. Figure 2C and D represents experiments showing that de novo made SynA, expressed from alcAp, is significantly co-localized with the late Golgi/TGN PHOSBP marker (PCC = 0.67, P < 0.0001), but only little with the early Golgi SedV marker (PCC = 0.37, P < 0.0001). These results suggest that SynA passes from the late Golgi/TGN, as might be expected (but never shown before in A. nidulans). On the other hand, we did not detect significant co-localization of cargoes (SynA or UapA) at the level of the early Golgi. Given that the presence of SynA at the TGN would, in principle, necessitate sorting from the early Golgi, this means that our methodology is not sensitive enough to detect transient passage from early secretory compartments. Thus, at this point, this technical limitation forced us to consider that a fraction of UapA might still be sorted at least to the early Golgi. Thus, we subsequently looked for more rigorous evidence supporting the suspected Golgi bypass of UapA trafficking. Localization of de novo made UapA in the PM in the absence of conventional secretion To obtain more compelling evidence for the apparent bypass of Golgi by UapA, we followed the trafficking of neosynthesized UapA-GFP in genetic backgrounds where distinct steps of the Golgi-dependent conventional secretory pathway were blocked for a defined period of time, via tight transcriptional repression of specific genes. For this, we constructed strains where the promoters of endogenous genes encoding SedVSed5 or GeaAGea1 (early/medial-Golgi), HypBSec7 (late-Golgi/TGN), RabERab11 (TGN/post-Golgi), AP-1σ (post-Golgi secretion) and ClaH (clathrin heavy chain/post-Golgi secretion) were replaced, through targeted homologous recombination, by the thiA promoter (thiAp). This promoter is tightly repressible upon addition of thiamine in the growth medium (Fig 3A). Western blot analysis showed that addition of thiamine to a culture of conidiospores of A. nidulans at the onset of incubation (ab initio) leads to dramatic reduction or absence of the relative protein after 12 h of germination (Fig 3B). At 12-14 h, when secretory proteins are not or hardly detected in Western blots, growth of germlings stops (Fig 3C) and the tip of A. nidulans swells in mutants lacking SedV, GeaA, RabE or ClaH, but less so in the case of HypB or AP-1 (Fig 3D). This dramatic morphological change in the apical part of growing hyphae is strong evidence that Golgi- and post-Golgi-dependent cargo trafficking is inhibited because cargo secretion in filamentous fungi is essential for maintaining polarity and growth. During establishment of repression of secretion, the transcription of UapA from its endogenous promoter is also kept fully repressed, using ammonium ions as sole N source 35. After establishment of repression and blocking of secretion (12- to 14-h germination), as evidenced by Western blot analysis, swelling of the tip and arrested growth, the transcription of UapA is derepressed essentially as previously described, via its native promoter. This system allowed us to follow UapA localization, while the conventional pathway is dramatically blocked at distinct Golgi-dependent steps. Figure 3. Localization of neosynthesized UapA in the PM when conventional secretion is blocked A. Schema depicting the strategy for blocking conventional secretion. B. Key endogenous genes controlling Golgi (sedV, geaA, hypB) or post-Golgi trafficking (rabE, ap1σ, claH) were genetically replaced by versions transcribed under the highly repressible thiAp promoter via targeted homologous recombination. In the absence of thiamine from the growth medium (derepressed conditions), the relative proteins are expressed, while upon addition of thiamine at the onset of conidiospore germination (ab initio repression), the expression of these proteins is tightly repressed. Proteins are detected by a standard Western blot analysis using either anti-FLAG or anti-GFP antibodies for Golgi and Post-Golgi proteins. Equal loading and protein steady-state levels are normalized against the amount of actin, d
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