@phdthesis{Tegtmeier2022,
  author    = {Tegtmeier, Laura},
  title     = {Functional analysis of ENTH domain proteins},
  doi       = {10.25932/publishup-57004},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-570049},
  school      = {Universit{\"a}t Potsdam},
  pages     = {106},
  year      = {2022},
  abstract  = {In plant cells, subcellular transport of cargo proteins relies to a large extent on post-Golgi transport pathways, many of which are mediated by clathrin-coated vesicles (CCVs). Vesicle formation is facilitated by different factors like accessory proteins and adaptor protein complexes (APs), the latter serving as a bridge between cargo proteins and the coat protein clathrin. One type of accessory proteins is defined by a conserved EPSIN N-TERMINAL HOMOLOGY (ENTH) domain and interacts with APs and clathrin via motifs in the C-terminal part. In Arabidopsis thaliana, there are three closely related ENTH domain proteins (EPSIN1, 2 and 3) and one highly conserved but phylogenetically distant outlier, termed MODIFIED TRANSPORT TO THE VACUOLE1 (MTV1). In case of the trans-Golgi network (TGN) located MTV1, clathrin association and a role in vacuolar transport have been shown previously (Sauer et al. 2013). In contrast, for EPSIN1 and EPSIN2 limited functional and localization data were available; and EPSIN3 remained completely uncharacterized prior to this study (Song et al. 2006; Lee et al. 2007). The molecular details of ENTH domain proteins in plants are still unknown. In order to systematically characterize all four ENTH proteins in planta, we first investigated expression and subcellular localization by analysis of stable reporter lines under their endogenous promotors. Although all four genes are ubiquitously expressed, their subcellular distribution differs markedly. EPSIN1 and MTV1 are located at the TGN, whereas EPSIN2 and EPSIN3 are associated with the plasma membrane (PM) and the cell plate. To examine potential functional redundancy, we isolated knockout T-DNA mutant lines and created all higher order mutant combinations. The clearest evidence for functional redundancy was observed in the epsin1 mtv1 double mutant, which is a dwarf displaying overall growth reduction. These findings are in line with the TGN localization of both MTV1 and EPS1. In contrast, loss of EPSIN2 and EPSIN3 does not result in a growth phenotype compared to wild type, however, a triple knockout of EPSIN1, EPSIN2 and EPSIN3 shows partially sterile plants. We focused mainly on the epsin1 mtv1 double mutant and addressed the functional role of these two genes in clathrin-mediated vesicle transport by comprehensive molecular, biochemical, and genetic analyses. Our results demonstrate that EPSIN1 and MTV1 promote vacuolar transport and secretion of a subset of cargo. However, they do not seem to be involved in endocytosis and recycling. Importantly, employing high-resolution imaging, genetic and biochemical experiments probing the relationship of the AP complexes, we found that EPSIN1/AP1 and MTV1/AP4 define two spatially and molecularly distinct subdomains of the TGN. The AP4 complex is essential for MTV1 recruitment to the TGN, whereas EPSIN1 is independent of AP4 but presumably acts in an AP1-dependent framework. Our findings suggest that this ENTH/AP pairing preference is conserved between animals and plants.},
  language  = {en}
}
@phdthesis{Freimuth2024,
  author    = {Freimuth, Nina},
  title     = {Elucidating the suppression of root hair formation by a member of a novel, short ENTH protein family in Arabidopsis thaliana},
  doi       = {10.25932/publishup-63499},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-634994},
  school      = {Universit{\"a}t Potsdam},
  pages     = {XIII, 156},
  year      = {2024},
  abstract  = {This work analyzed functional and regulatory aspects of the so far little characterized EPSIN N-terminal Homology (ENTH) domain-containing protein EPSINOID2 in Arabidopsis thaliana. ENTH domain proteins play accessory roles in the formation of clathrin-coated vesicles (CCVs) (Zouhar and Sauer 2014). Their ENTH domain interacts with membranes and their typically long, unstructured C-terminus contains binding motifs for adaptor protein complexes and clathrin itself. There are seven ENTH domain proteins in Arabidopsis. Four of them possess the canonical long C-terminus and participate in various, presumably CCV-related intracellular transport processes (Song et al. 2006; Lee et al. 2007; Sauer et al. 2013; Collins et al. 2020; Heinze et al. 2020; Mason et al. 2023). The remaining three ENTH domain proteins, however, have severely truncated C-termini and were termed EPSINOIDs (Zouhar and Sauer 2014; Freimuth 2015). Their functions are currently unclear. Preceding studies focusing on EPSINOID2 indicated a role in root hair formation: epsinoid2 T DNA mutants exhibited an increased root hair density and EPSINOID2-GFP was specifically located in non-hair cell files in the Arabidopsis root epidermis (Freimuth 2015, 2019). In this work, it was clearly shown that loss of EPSINOID2 leads to an increase in root hair density through analyses of three independent mutant alleles, including a newly generated CRISPR/Cas9 full deletion mutant. The ectopic root hairs emerging from non-hair positions in all epsinoid2 mutant alleles are most likely not a consequence of altered cell fate, because extensive genetic analyses placed EPSINOID2 downstream of the established epidermal patterning network. Thus, EPSINOID2 seems to act as a cell autonomous inhibitor of root hair formation. Attempts to confirm this hypothesis by ectopically overexpressing EPSINOID2 led to the discovery of post-transcriptional and -translational regulation through different mechanisms. One involves the little characterized miRNA844-3p. Interference with this pathway resulted in ectopic EPSINOID2 overexpression and decreased root hair density, confirming it as negative factor in root hair formation. A second mechanism likely involves proteasomal degradation. Treatment with proteasomal inhibitor MG132 led to EPSINOID2-GFP accumulation, and a KEN box degron motif was identified in the EPSINOID2 sequence associated with degradation through a ubiquitin/proteasome-dependent pathway. In line with a tight dose regulation, genetic analyses of all three mutant alleles indicate that EPSINOID2 is haploinsufficient. Lastly, it was revealed that, although EPSINOID2 promoter activity was found in all epidermal cells, protein accumulation was observed in N-cells only, hinting at yet another layer of regulation.},
  language  = {en}
}