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AP-2 is critical for ergosterol membrane localization. (A) Apical filipin staining of ergosterol in hyphal cells of wild-type and mutants. Notice the significant alterations and loss or reduction in apical staining in ap2 s D, thiA p-basA, thiA p-slaB and stoAD genetic backgrounds. Representative phenotypes selected from n = 20 hyphae for wt and mutant strains. Biological replicates: 2, Technical replicates: 20. Scale bars represent 5 mm. (B) Quantitative analysis of fluorescence intensity of filipin staining along the surface of hyphae tips. The region measured is depicted in the cartoon on the left. DOI: 10.7554/eLife.20083.020 

AP-2 is critical for ergosterol membrane localization. (A) Apical filipin staining of ergosterol in hyphal cells of wild-type and mutants. Notice the significant alterations and loss or reduction in apical staining in ap2 s D, thiA p-basA, thiA p-slaB and stoAD genetic backgrounds. Representative phenotypes selected from n = 20 hyphae for wt and mutant strains. Biological replicates: 2, Technical replicates: 20. Scale bars represent 5 mm. (B) Quantitative analysis of fluorescence intensity of filipin staining along the surface of hyphae tips. The region measured is depicted in the cartoon on the left. DOI: 10.7554/eLife.20083.020 

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Article
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Filamentous fungi provide excellent systems for investigating the role of the AP-2 complex in polar growth. Using Aspergillus nidulans, we show that AP-2 has a clathrin-independent essential role in polarity maintenance and growth. This is in line with a sequence analysis showing that the AP-2 b subunit (b2) of higher fungi lacks a clathrin-binding...

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Context 1
... observed significant depolarization, often associated with the appearance of discrete cortical foci and abnormal filipin staining in the ap2 s D, as well as, in thiA p -slaB, stoAD and thiA p -basA mutant backgrounds. In contrast, we observed a polar localization of filipin in thiA p -claL, dnfAD, dnfBD and sagAD mutant strains, similar to wild-type ( Figure 9A). Quantification of these results, by measuring the strength of the filipin fluorescent signal along the tip, confirmed the critical role of AP-2, as well as, of BasA, SlaB or StoA, in the apical depositioning of ergosterol ( Figure 9B). ...
Context 2
... contrast, we observed a polar localization of filipin in thiA p -claL, dnfAD, dnfBD and sagAD mutant strains, similar to wild-type ( Figure 9A). Quantification of these results, by measuring the strength of the filipin fluorescent signal along the tip, confirmed the critical role of AP-2, as well as, of BasA, SlaB or StoA, in the apical depositioning of ergosterol ( Figure 9B). These results further supported that AP-2 has a specific role in lipid apical localization, distinct from clathrin, necessary polar growth. ...
Context 3
... observed significant depolarization, often associated with the appearance of discrete cortical foci and abnormal filipin staining in the ap2 s D, as well as, in thiA p -slaB, stoAD and thiA p -basA mutant backgrounds. In contrast, we observed a polar localization of filipin in thiA p -claL, dnfAD, dnfBD and sagAD mutant strains, similar to wild-type ( Figure 9A). Quantification of these results, by measuring the strength of the filipin fluorescent signal along the tip, confirmed the critical role of AP-2, as well as, of BasA, SlaB or StoA, in the apical depositioning of ergosterol ( Figure 9B). ...
Context 4
... contrast, we observed a polar localization of filipin in thiA p -claL, dnfAD, dnfBD and sagAD mutant strains, similar to wild-type ( Figure 9A). Quantification of these results, by measuring the strength of the filipin fluorescent signal along the tip, confirmed the critical role of AP-2, as well as, of BasA, SlaB or StoA, in the apical depositioning of ergosterol ( Figure 9B). These results further supported that AP-2 has a specific role in lipid apical localization, distinct from clathrin, necessary polar growth. ...
Context 5
... observed significant depolarization, often associated with the appearance of discrete cortical foci and abnormal filipin staining in the ap2 s D, as well as, in thiA p -slaB, stoAD and thiA p -basA mutant backgrounds. In contrast, we observed a polar localization of filipin in thiA p -claL, dnfAD, dnfBD and sagAD mutant strains, similar to wild-type ( Figure 9A). Quantification of these results, by measuring the strength of the filipin fluorescent signal along the tip, confirmed the critical role of AP-2, as well as, of BasA, SlaB or StoA, in the apical depositioning of ergosterol ( Figure 9B). ...
Context 6
... contrast, we observed a polar localization of filipin in thiA p -claL, dnfAD, dnfBD and sagAD mutant strains, similar to wild-type ( Figure 9A). Quantification of these results, by measuring the strength of the filipin fluorescent signal along the tip, confirmed the critical role of AP-2, as well as, of BasA, SlaB or StoA, in the apical depositioning of ergosterol ( Figure 9B). These results further supported that AP-2 has a specific role in lipid apical localization, distinct from clathrin, necessary polar growth. ...

Citations

... Golgi-bypass mechanism (Sánchez-León et al, 2011 these studies concern trafficking routes operating only under specific conditions or 764 stress, and do not seem to reflect a major trafficking mechanism for the biogenesis of 765 essential cargoes, as transporters or receptors, proposed through our work. Notice also 766 that in A. nidulans transporters and apical cargoes use different endocytic mechanisms 767 for their internalization, turnover or recycling (Martzoukou et al, 2017). 768 ...
Article
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Membrane proteins are sorted to the plasma membrane (PM) via Golgi-dependent trafficking. However, our recent studies challenged the essentiality of Golgi in the biogenesis of specific transporters. Here, we investigate the trafficking mechanisms of membrane proteins by following the localization of the polarized R-SNARE SynA versus the non-polarized transporter UapA, 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 finding that, unlike SynA, UapA does not colocalize with the late-Golgi. In line with early partitioning into distinct secretory carriers, the two cargoes collapse in distinct ERES in a sec31 ts background. Trafficking via distinct cargo-specific carriers is further supported by showing that repression of proteins essential for conventional cargo secretion does not affect UapA trafficking, while blocking SynA secretion. Overall, this work establishes the existence of distinct, cargo-dependent, trafficking mechanisms, initiating at ERES and being differently dependent on Golgi and SNARE interactions.
... The subcellular localization of AnLsb6 shows the characteristic polarized distribution of vesicular structures (Golgi cisternae), resembling localization of other characterized Golgi-associated proteins in A. nidulans 45,46 . For this reason, colocalization was attempted using available established markers for the early and late Golgi compartments, such as the early Golgi t-SNARE SedV Sed5 , the pleckstrin homology domain of the oxysterol binding protein (PH OSBP ) binding on Golgi PtdIns4P enriched membranes (see also later), the Ras GTPase ArfA ARF1 involved in Golgi organization and secretion 47,48 , the HypB SEC7 Arf1 GEF 45,49,50 , or the clathrin heavy chain ClaH coating secretory vesicles at the post Golgi 51,52 . This analysis showed that whereas less colocalization is observed with the early Golgi marker SedV SED5 53 , a significant association of GFP-AnLsb6 is observed with all other late Golgi markers ( Fig. 2a and Supplementary Data 2). ...
... This analysis showed that whereas less colocalization is observed with the early Golgi marker SedV SED5 53 , a significant association of GFP-AnLsb6 is observed with all other late Golgi markers ( Fig. 2a and Supplementary Data 2). This association with later Golgi stages is further supported by chase experiments in the presence of the Golgi inhibitor Brefeldin A, where preferentially GFP-AnLsb6 and PH OSBP tend to colocalize in transiently collapsing Brefeldin bodies (Fig. 2b) [52][53][54] , by the mis-localization of GFP-AnLsb6 towards the hyphal apex when the trans-Golgi Arf1 GEF HypB SEC7 is depleted, by the strong dis-organization and appearance of cytoplasmic haze when clathrin heavy chain is depleted 52,53,55 , the latter also suggesting that GFP-AnLsb6 may also be operating more downstream at the late/post Golgi stage ( Supplementary Fig. 3a), and finally by the effective reconstitution of bimolecular fluorescence (BiFC) in strains carrying AnLsb6 and HypB SEC7 tagged with N-terminal YFP N and C-terminal YFP C , respectively, indicating at least close proximity of these two proteins ( Supplementary Fig. 3b). ...
... This analysis showed that whereas less colocalization is observed with the early Golgi marker SedV SED5 53 , a significant association of GFP-AnLsb6 is observed with all other late Golgi markers ( Fig. 2a and Supplementary Data 2). This association with later Golgi stages is further supported by chase experiments in the presence of the Golgi inhibitor Brefeldin A, where preferentially GFP-AnLsb6 and PH OSBP tend to colocalize in transiently collapsing Brefeldin bodies (Fig. 2b) [52][53][54] , by the mis-localization of GFP-AnLsb6 towards the hyphal apex when the trans-Golgi Arf1 GEF HypB SEC7 is depleted, by the strong dis-organization and appearance of cytoplasmic haze when clathrin heavy chain is depleted 52,53,55 , the latter also suggesting that GFP-AnLsb6 may also be operating more downstream at the late/post Golgi stage ( Supplementary Fig. 3a), and finally by the effective reconstitution of bimolecular fluorescence (BiFC) in strains carrying AnLsb6 and HypB SEC7 tagged with N-terminal YFP N and C-terminal YFP C , respectively, indicating at least close proximity of these two proteins ( Supplementary Fig. 3b). ...
Article
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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.
... However, most of these studies concern trafficking routes operating only under specific conditions or stress, and do not seem to reflect a major trafficking mechanism for the biogenesis of essential cargoes, as transporters or receptors, proposed through our work. Notice also that in A. nidulans transporters and apical cargoes use different endocytic mechanisms for their internalization, turnover or recycling (Martzoukou et al, 2017). ...
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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 fungus Aspergillus nidulans challenged 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.
... Samples were prepared as previously described (Martzoukou et al., 2017). Unless otherwise stated, conidiospores were incubated overnight in glass bottom 35-mm l-dishes (Ibidi, Lab Supplies Scientific SA, Hellas) in liquid minimal media, for 16-22 h at 25 • C, under conditions of transcriptional repression of the studied membrane cargoes, UapA and SynA . ...
... Images were further processed and annotated in Adobe Photoshop CS4 Extended version 11.0.2. Technical replicates correspond to different hyphal cells observed within each sample, while biological replicates correspond to different samples (Martzoukou et al., 2017). For quantifying co-localization, Pearson's correlation coefficient (PCC) above thresholds, for a selected region of interest (ROI), was calculated using the ICY co-localization studio plugin (pixel-based method) (http s://icy.bioimageanalysis.org/). ...
... Secretion dynamics of AnChsB were shown to occur at hyphal apical regions via indirect endocytic recycling, where the diffused protein is internalized by subapical actin patches (an endocytosis site) and reconducted from endosomes to the trans-Golgi network cisternae in a Sec7-, GARP-(Golgi-associated retrograde protein) and Rab6-dependent manner [126]. In addition, the adaptor protein (AP)-2 complex, which is involved in the endocytosis process [127], promotes AnChsB internalization from the subapical collars of the hyphal surface, and is also important for AnChsB localization at hyphal tips [128]. ...
Article
Full-text available
The fungal cell wall (FCW) is a dynamic structure responsible for the maintenance of cellular homeostasis, and is essential for modulating the interaction of the fungus with its environment. It is composed of proteins, lipids, pigments and polysaccharides, including chitin. Chitin synthesis is catalyzed by chitin synthases (CS), and up to eight CS-encoding genes can be found in Aspergillus species. This review discusses in detail the chitin synthesis and regulation in Aspergillus species, and how manipulation of chitin synthesis pathways can modulate fungal growth, enzyme production, virulence and susceptibility to antifungal agents. More specifically, the metabolic steps involved in chitin biosynthesis are described with an emphasis on how the initiation of chitin biosynthesis remains unknown. A description of the classification, localization and transport of CS was also made. Chitin biosynthesis is shown to underlie a complex regulatory network, with extensive cross-talks existing between the different signaling pathways. Furthermore, pathways and recently identified regulators of chitin biosynthesis during the caspofungin paradoxical effect (CPE) are described. The effect of a chitin on the mammalian immune system is also discussed. Lastly, interference with chitin biosynthesis may also be beneficial for biotechnological applications. Even after more than 30 years of research, chitin biosynthesis remains a topic of current interest in mycology.
... Like the function of the FgAP-2 complex in F. graminearum (41,42), the FonAP-2 complex is essential for vegetative growth and hyphal morphology, conidiation and conidial morphology, cell wall integrity, and virulence in Fon. In particularly, disruption of each of the FonAP-2 complex subunits resulted in defects in hyphal polarized growth and cell wall stress response, which is in agreement with the observations that the AP-2 complex is involved in regulating cell wall integrity in F. graminearum and yeast (40,42). The facts that FonPATs were involved in oxidative stress response and that the FonAP-2 complex subunits and their palmitoylation are critical to the cell wall integrity may imply a relationship among the FonPAT2-mediated palmitoylation of FonAP-2 complex subunits, cell wall integrity, and oxidative stress response in Fon. ...
Article
Full-text available
Fusarium oxysporum f. sp. niveum ( Fon ), the causal agent of watermelon Fusarium wilt, is one of the most serious threats for the sustainable development of the watermelon industry worldwide. However, little is known about the underlying molecular mechanism of pathogenicity in Fon .
... The absence of TAT-5, the nematode (Caenorhabditis elegans) homolog of Drs2, results in considerable shedding of plasma membrane-derived extracellular vesicles (Wehman et al., 2011;Beer et al., 2018). P4-ATPase also participates in coat protein complex I- (Hua et al., 2002;Xu et al., 2017), clathrin-, adaptor- (Chen et al., 1999;Liu et al., 2008;Schultzhaus et al., 2015;Martzoukou et al., 2017), and retromer-dependent trafficking routes (Wu et al., 2016;Dalton et al., 2017;McGough et al., 2018). ...
Article
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Pollen tube guidance regulates the growth direction and ovule targeting of pollen tubes in pistils, which is crucial for the completion of sexual reproduction in flowering plants. The Arabidopsis (Arabidopsis thaliana) pollen-specific receptor kinase (PRK) family members PRK3 and PRK6 are specifically tip-localized and essential for pollen tube growth and guidance. However, the mechanisms controlling the polar localization of PRKs at the pollen tube tip are unclear. The Arabidopsis P4-ATPase ALA3 helps establish the polar localization of apical phosphatidylserine (PS) in pollen tubes. Here, we discovered that loss of ALA3 function caused pollen tube defects in growth and ovule targeting and significantly affected the polar localization pattern of PRK3 and PRK6. Both PRK3 and PRK6 contain two polybasic clusters in the intracellular juxtamembrane domain, and they bound to PS in vitro. PRK3 and PRK6 with polybasic cluster mutations showed reduced or abolished binding to PS and altered polar localization patterns, and they failed to effectively complement the pollen tube-related phenotypes of prk mutants. These results suggest that ALA3 influences the precise localization of PRK3, PRK6 and other PRKs by regulating the distribution of PS, which plays a key role in regulating pollen tube growth and guidance.
... For example, elimination of the AP2b subunits causes only a mild reduction in the endocytic internalization of the transferrin receptor, due to the substitution of AP2b with AP1b (Keyel et al., 2008). Only fungi seems to have noninterchangeable b subunits for AP-1 and AP-2 (Dacks et al., 2008); however, the fungal b subunits of both AP-1 and AP-2 lack the canonical clathrin-binding domains and AP-2 has a clathrin-independent role in endocytosis in filamentous fungi (Martzoukou et al., 2017), suggesting that the fungal AP-1 and AP-2 complexes may have evolved divergent functions. We have found that the two putative AP1/2b subunits in Arabidopsis (b1 and b2) are able to co-assemble with subunits of both AP-1 and AP-2 (Figure 1), and consistently, localize to both TGN and plasma membrane (Figure 3, A and C), the cognate membranes for each complex. ...
Article
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AP-1 and AP-2 adaptor protein complexes mediate clathrin-dependent trafficking at the trans-Golgi network (TGN) and the plasma membrane, respectively. Whereas AP-1 is required for trafficking to plasma membrane and vacuoles, AP-2 mediates endocytosis. These AP complexes consist of four subunits (adaptins): two large subunits (β1 and γ for AP-1 and β2 and α for AP-2), a medium subunit μ, and a small subunit σ. In general, adaptins are unique to each AP complex, with the exception of β subunits that are shared by AP-1 and AP-2 in some invertebrates. Here, we show that the two putative Arabidopsis thaliana AP1/2β adaptins co-assemble with both AP-1 and AP-2 subunits and regulate exocytosis and endocytosis in root cells, consistent with their dual localization at the TGN and plasma membrane. Deletion of both β adaptins is lethal in plants. We identified a critical role of β adaptins in pollen wall formation and reproduction, involving the regulation of membrane trafficking in the tapetum and pollen germination. In tapetal cells, β adaptins localize almost exclusively to the TGN and mediate exocytosis of the plasma membrane transporters such as ABCG9 and ABCG16. This study highlights the essential role of AP1/2β adaptins in plants and their specialized roles in specific cell types.
... The images were further processed and annotated in Adobe Photoshop CS4 Extended version 11.0.2. Technical replicates correspond to different hyphal cells observed within each sample, while biological replicates correspond to different samples (Martzoukou et al., 2017). For quantifying co-localization (Dunn et al., 2011), Pearson's correlation coefficient (PCC) above thresholds, for a selected region of interest (ROI), was calculated using the ICY co-localization studio plugin (pixel-based method) (http://icy.bioimageanalysis.org/). ...
Article
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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.
... From a more general perspective our findings dissent from the widely held view that AP-2 obligatorily associates with clathrin to execute its cell physiological functions, at least in central nervous system (CNS) neurons. While the most well-known function of AP-2 is its involvement in CME in mammalian cells and tissues, studies in higher fungi have uncovered a clathrin-independent role of fungal AP-2 in the polar localization of the lipid flippases DnfA and DnfB (Martzoukou et al., 2017). Interestingly, in this system AP-2 is seen to colocalize with endocytic markers and the actin-associated protein AbpA, but not with clathrin (Martzoukou et al., 2017). ...
... While the most well-known function of AP-2 is its involvement in CME in mammalian cells and tissues, studies in higher fungi have uncovered a clathrin-independent role of fungal AP-2 in the polar localization of the lipid flippases DnfA and DnfB (Martzoukou et al., 2017). Interestingly, in this system AP-2 is seen to colocalize with endocytic markers and the actin-associated protein AbpA, but not with clathrin (Martzoukou et al., 2017). Because AP-2 also colocalizes with a fungal homolog of Synaptobrevin (Martzoukou et al., 2017), and clathrin-independent SV endocytosis at hippocampal synapses depends on actin polymerization (Soykan et al., 2017), our newly observed function of AP-2 might reflect an unexpectedly widely conserved endocytic mechanism. ...
... Interestingly, in this system AP-2 is seen to colocalize with endocytic markers and the actin-associated protein AbpA, but not with clathrin (Martzoukou et al., 2017). Because AP-2 also colocalizes with a fungal homolog of Synaptobrevin (Martzoukou et al., 2017), and clathrin-independent SV endocytosis at hippocampal synapses depends on actin polymerization (Soykan et al., 2017), our newly observed function of AP-2 might reflect an unexpectedly widely conserved endocytic mechanism. Conversely, studies in AP-2 KO cells have revealed AP-2-independent forms of CME in mammals that impact on receptor sorting and signaling (Pascolutti et al., 2019). ...
Article
Full-text available
Neurotransmission is based on the exocytic fusion of synaptic vesicles (SVs) followed by endocytic membrane retrieval and the reformation of SVs. Conflicting models have been proposed regarding the mechanisms of SV endocytosis, most notably clathrin/adaptor protein complex 2 (AP-2)-mediated endocytosis and clathrin-independent ultrafast endocytosis. Partitioning between these pathways has been suggested to be controlled by temperature and stimulus paradigm. We report on the comprehensive survey of six major SV proteins to show that SV endocytosis in mouse hippocampal neurons at physiological temperature occurs independent of clathrin while the endocytic retrieval of a subset of SV proteins including the vesicular transporters for glutamate and GABA depend on sorting by the clathrin adaptor AP-2. Our findings highlight a clathrin-independent role of the clathrin adaptor AP-2 in the endocytic retrieval of select SV cargos from the presynaptic cell surface and suggest a revised model for the endocytosis of SV membranes at mammalian central synapses.