Early endosomes sort transmembrane cargo either for lysosomal degradation or retrieval to the plasma membrane or the Golgi complex. Endosomal retrieval in eukaryotes is governed by the anciently homologous retromer or retriever complexes. Each comprises a core tri-protein subcomplex, membrane-deformation proteins and interacting partner complexes, together retrieving a variety of known cargo proteins. Trichomonas vaginalis, a sexually transmitted human parasite, uses the endomembrane system for pathogenesis. It has massively and selectively expanded its endomembrane protein complement, the evolutionary path of which has been largely unexplored. Our molecular evolutionary study of retromer, retriever and associated machinery in parabasalids and its free-living sister lineage of Anaeramoeba demonstrates specific expansion of the retromer machinery, contrasting with the retriever components. We also observed partial loss of the Commander complex and sorting nexins in Parabasalia but complete retention in Anaeramoeba. Notably, we identified putative parabasalid sorting nexin analogs. Finally, we report the first retriever protein localization in a non-metazoan group along with retromer protein localization in T. vaginalis.

• Klíčová slova
• Endomembrane, Evolution, Parabasalids, Phylogenomics, Retriever, Retromer,
• MeSH
• endozomy * metabolismus   MeSH
• fylogeneze MeSH
• Golgiho aparát metabolismus   MeSH
• lidé MeSH
• molekulární evoluce MeSH
• protozoální proteiny metabolismus   genetika   MeSH
• transport proteinů MeSH
• Trichomonas vaginalis metabolismus   genetika   MeSH
• třídící nexiny metabolismus   genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• protozoální proteiny MeSH
• třídící nexiny MeSH

The pentameric WASH complex facilitates endosomal protein sorting by activating Arp2/3, which in turn leads to the formation of F-actin patches specifically on the endosomal surface. It is generally accepted that WASH complex attaches to the endosomal membrane via the interaction of its subunit FAM21 with the retromer subunit VPS35. However, we observe the WASH complex and F-actin present on endosomes even in the absence of VPS35. We show that the WASH complex binds to the endosomal surface in both a retromer-dependent and a retromer-independent manner. The retromer-independent membrane anchor is directly mediated by the subunit SWIP. Furthermore, SWIP can interact with a number of phosphoinositide species. Of those, our data suggest that the interaction with phosphatidylinositol-3,5-bisphosphate (PI(3,5)P2 ) is crucial to the endosomal binding of SWIP. Overall, this study reveals a new role of the WASH complex subunit SWIP and highlights the WASH complex as an independent, self-sufficient trafficking regulator.

• Klíčová slova
• SWIP, VPS35, WASH complex, endosome, phosphatidylinositol-3,5-bisphosphate, retromer,
• MeSH
• aktiny * metabolismus   MeSH
• endozomy metabolismus   MeSH
• intracelulární signální peptidy a proteiny * metabolismus   MeSH
• lidé MeSH
• mikrofilamentové proteiny metabolismus   MeSH
• transport proteinů MeSH
• vezikulární transportní proteiny * metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aktiny * MeSH
• intracelulární signální peptidy a proteiny * MeSH
• mikrofilamentové proteiny MeSH
• vezikulární transportní proteiny * MeSH

Intracellular protein routing is mediated by vesicular transport which is tightly regulated in eukaryotes. The protein and lipid homeostasis depends on coordinated delivery of de novo synthesized or recycled cargoes to the plasma membrane by exocytosis and their subsequent removal by rerouting them for recycling or degradation. Here, we report the characterization of protein affected trafficking 3 (pat3) mutant that we identified by an epifluorescence-based forward genetic screen for mutants defective in subcellular distribution of Arabidopsis auxin transporter PIN1-GFP. While pat3 displays largely normal plant morphology and development in nutrient-rich conditions, it shows strong ectopic intracellular accumulations of different plasma membrane cargoes in structures that resemble prevacuolar compartments (PVC) with an aberrant morphology. Genetic mapping revealed that pat3 is defective in vacuolar protein sorting 35A (VPS35A), a putative subunit of the retromer complex that mediates retrograde trafficking between the PVC and trans-Golgi network. Similarly, a mutant defective in another retromer subunit, vps29, shows comparable subcellular defects in PVC morphology and protein accumulation. Thus, our data provide evidence that the retromer components VPS35A and VPS29 are essential for normal PVC morphology and normal trafficking of plasma membrane proteins in plants. In addition, we show that, out of the three VPS35 retromer subunits present in Arabidopsis thaliana genome, the VPS35 homolog A plays a prevailing role in trafficking to the lytic vacuole, presenting another level of complexity in the retromer-dependent vacuolar sorting.

• Klíčová slova
• Arabidopsis thaliana., VPS29, VPS35, prevacuolar compartment (PVC), retromer, vacuolar trafficking,
• MeSH
• Arabidopsis genetika   fyziologie   MeSH
• endocytóza MeSH
• kompartmentace buňky * MeSH
• membránové proteiny metabolismus   fyziologie   MeSH
• molekulární sekvence - údaje MeSH
• mutace MeSH
• proteiny huseníčku metabolismus   fyziologie   MeSH
• transport proteinů MeSH
• vakuoly metabolismus   MeSH
• vezikulární transportní proteiny metabolismus   fyziologie   MeSH
• zelené fluorescenční proteiny genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• Mag1 protein, Arabidopsis MeSH Prohlížeč
• membránové proteiny MeSH
• proteiny huseníčku MeSH
• vezikulární transportní proteiny MeSH
• VPS35 protein, Arabidopsis MeSH Prohlížeč
• zelené fluorescenční proteiny MeSH

Vacuolar proteins play essential roles in plant physiology and development, but the factors and the machinery regulating their vesicle trafficking through the endomembrane compartments remain largely unknown. We and others have recently identified an evolutionarily conserved plant endosomal sorting complex required for transport (ESCRT)-associated protein apoptosis-linked gene-2 interacting protein X (ALIX), which plays canonical functions in the biogenesis of the multivesicular body/prevacuolar compartment (MVB/PVC) and in the sorting of ubiquitinated membrane proteins. In this study, we elucidate the roles and underlying mechanism of ALIX in regulating vacuolar transport of soluble proteins, beyond its conventional ESCRT function in eukaryotic cells. We show that ALIX colocalizes and physically interacts with the retromer core subunits Vps26 and Vps29 in planta. Moreover, double-mutant analysis reveals the genetic interaction of ALIX with Vps26 and Vps29 for regulating trafficking of soluble vacuolar proteins. Interestingly, depletion of ALIX perturbs membrane recruitment of Vps26 and Vps29 and alters the endosomal localization of vacuolar sorting receptors (VSRs). Taken together, ALIX functions as a unique retromer core subcomplex regulator by orchestrating receptor-mediated vacuolar sorting of soluble proteins.

• Klíčová slova
• ESCRT machiner, endosomal recycling, multivesicular body/prevacuolar compartment (MVB/PVC), retromer complex, vacuolar trafficking,
• MeSH
• Arabidopsis * metabolismus   MeSH
• endozomální třídící komplexy pro transport metabolismus   MeSH
• endozomy metabolismus   MeSH
• proteiny huseníčku * genetika   metabolismus   MeSH
• rostliny metabolismus   MeSH
• transport proteinů fyziologie   MeSH
• transportní proteiny metabolismus   MeSH
• vakuoly metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ALIX protein, Arabidopsis MeSH Prohlížeč
• endozomální třídící komplexy pro transport MeSH
• proteiny huseníčku * MeSH
• transportní proteiny MeSH

The plant hormone gibberellic acid (GA) is a crucial regulator of growth and development. The main paradigm of GA signaling puts forward transcriptional regulation via the degradation of DELLA transcriptional repressors. GA has also been shown to regulate tropic responses by modulation of the plasma membrane incidence of PIN auxin transporters by an unclear mechanism. Here we uncovered the cellular and molecular mechanisms by which GA redirects protein trafficking and thus regulates cell surface functionality. Photoconvertible reporters revealed that GA balances the protein traffic between the vacuole degradation route and recycling back to the cell surface. Low GA levels promote vacuolar delivery and degradation of multiple cargos, including PIN proteins, whereas high GA levels promote their recycling to the plasma membrane. This GA effect requires components of the retromer complex, such as Sorting Nexin 1 (SNX1) and its interacting, microtubule (MT)-associated protein, the Cytoplasmic Linker-Associated Protein (CLASP1). Accordingly, GA regulates the subcellular distribution of SNX1 and CLASP1, and the intact MT cytoskeleton is essential for the GA effect on trafficking. This GA cellular action occurs through DELLA proteins that regulate the MT and retromer presumably via their interaction partners Prefoldins (PFDs). Our study identified a branching of the GA signaling pathway at the level of DELLA proteins, which, in parallel to regulating transcription, also target by a nontranscriptional mechanism the retromer complex acting at the intersection of the degradation and recycling trafficking routes. By this mechanism, GA can redirect receptors and transporters to the cell surface, thus coregulating multiple processes, including PIN-dependent auxin fluxes during tropic responses.

• Klíčová slova
• DELLA, gibberellin, microtubules, polar auxin transport, vesicle trafficking,
• MeSH
• Arabidopsis růst a vývoj   metabolismus   MeSH
• buněčná membrána metabolismus   MeSH
• gibereliny farmakologie   MeSH
• kyseliny indoloctové farmakologie   MeSH
• mikrotubuly metabolismus   MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• regulace genové exprese u rostlin účinky léků   MeSH
• regulátory růstu rostlin farmakologie   MeSH
• signální transdukce MeSH
• transport proteinů MeSH
• třídící nexiny genetika   metabolismus   MeSH
• vakuoly metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• GAI protein, Arabidopsis MeSH Prohlížeč
• gibereliny MeSH
• kyseliny indoloctové MeSH
• proteiny huseníčku MeSH
• regulátory růstu rostlin MeSH
• SNX1 protein, Arabidopsis MeSH Prohlížeč
• třídící nexiny MeSH

During Caenorhabditis elegans development, multiple cells migrate long distances or extend processes to reach their final position and/or attain proper shape. The Wnt signalling pathway stands out as one of the major coordinators of cell migration or cell outgrowth along the anterior-posterior body axis. The outcome of Wnt signalling is fine-tuned by various mechanisms including endocytosis. In this study, we show that SEL-5, the C. elegans orthologue of mammalian AP2-associated kinase AAK1, acts together with the retromer complex as a positive regulator of EGL-20/Wnt signalling during the migration of QL neuroblast daughter cells. At the same time, SEL-5 in cooperation with the retromer complex is also required during excretory canal cell outgrowth. Importantly, SEL-5 kinase activity is not required for its role in neuronal migration or excretory cell outgrowth, and neither of these processes is dependent on DPY-23/AP2M1 phosphorylation. We further establish that the Wnt proteins CWN-1 and CWN-2, together with the Frizzled receptor CFZ-2, positively regulate excretory cell outgrowth, while LIN-44/Wnt and LIN-17/Frizzled together generate a stop signal inhibiting its extension.

• Klíčová slova
• C. elegans, DPY-23/AP2M1, SEL-5/AAK1, Wnt signalling, cell biology, developmental biology, excretory cell, retromer,
• MeSH
• Caenorhabditis elegans * genetika   metabolismus   MeSH
• frizzled receptory metabolismus   genetika   MeSH
• pohyb buněk * MeSH
• proteiny Caenorhabditis elegans * metabolismus   genetika   MeSH
• proteiny Wnt metabolismus   genetika   MeSH
• signální dráha Wnt * MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• CWN-1 protein, C elegans MeSH Prohlížeč
• CWN-2 protein, C elegans MeSH Prohlížeč
• Egl-20 protein, C elegans MeSH Prohlížeč
• frizzled receptory MeSH
• proteiny Caenorhabditis elegans * MeSH
• proteiny Wnt MeSH

Truncating genetic variants of SORL1, encoding the endosome recycling receptor SORLA, have been accepted as causal of Alzheimer's disease (AD). However, most genetic variants observed in SORL1 are missense variants, for which it is complicated to determine the pathogenicity level because carriers come from pedigrees too small to be informative for penetrance estimations. Here, we describe three unrelated families in which the SORL1 coding missense variant rs772677709, that leads to a p.Y1816C substitution, segregates with Alzheimer's disease. Further, we investigate the effect of SORLA p.Y1816C on receptor maturation, cellular localization, and trafficking in cell-based assays. Under physiological circumstances, SORLA dimerizes within the endosome, allowing retromer-dependent trafficking from the endosome to the cell surface, where the luminal part is shed into the extracellular space (sSORLA). Our results showed that the p.Y1816C mutant impairs SORLA homodimerization in the endosome, leading to decreased trafficking to the cell surface and less sSORLA shedding. These trafficking defects of the mutant receptor can be rescued by the expression of the SORLA 3Fn-minireceptor. Finally, we find that iPSC-derived neurons with the engineered p.Y1816C mutation have enlarged endosomes, a defining cytopathology of AD. Our studies provide genetic as well as functional evidence that the SORL1 p.Y1816C variant is causal for AD. The partial penetrance of the mutation suggests this mutation should be considered in clinical genetic screening of multiplex early-onset AD families.

• Klíčová slova
• 3Fn-domain, SORL1-associated Alzheimer’s disease, SORLA, dimerization, retromer,
• MeSH
• Alzheimerova nemoc * genetika   metabolismus   patologie   MeSH
• endozomy * metabolismus   MeSH
• HEK293 buňky MeSH
• lidé středního věku MeSH
• lidé MeSH
• membránové transportní proteiny * genetika   metabolismus   MeSH
• missense mutace MeSH
• multimerizace proteinu MeSH
• proteiny související s LDL-receptory * genetika   metabolismus   MeSH
• rodokmen * MeSH
• senioři MeSH
• transport proteinů MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• membránové transportní proteiny * MeSH
• proteiny související s LDL-receptory * MeSH
• SORL1 protein, human MeSH Prohlížeč