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Lipid binding assay and subcellular localization of plant FYVE domain-containing protein FREE1. (a) Sequence alignment of FYVE domains from FREE1, HsEEA1, HsHrs, and ScFab1p proteins. (b) His-Sumo-FREE1 protein was expressed in E. coli, purified using a His SpinTrap column, and stained with Coomassie blue after separation by SDS-PAGE. M stands for protein maker; asterisk indicates purified protein. (c) Purified His-Sumo-FREE1 protein was used in an in vitro lipid-binding assay, followed by immunodetection with an anti-His antibody. (d) Colocalization of GFP-FREE1 with the MVB marker mRFP-Rha1 in the transgenic Arabidopsis root cells. Wortmannin treatment caused the enlargement of MVB to form ring-like structures (arrows). Localization of GFP-FREE1 on the membrane of enlarged MVB induced by wortmannin treatment indicates that the PI3P lipid binding is dispensable for the MVB localization of GFP-FREE1. Bars are 10 μm
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The FYVE domain is a double zinc finger-like domain that predominantly binds phosphatidylinositol 3-phosphate. The FYVE domain is usually found in proteins primarily involved in regulating various aspects of endomembrane homeostasis, including endosome tethering, endocytic recycling, membrane protein sorting, and autophagosome maturation. Whereas F...
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Context 1
... containing an FYVE domain, named after the first letter of the four proteins in which this domain was originally discovered: Fab1, YOTB, Vac1, and EEA1. The basic FYVE domain contains eight Zn 2+ coordinating cysteines, which are surrounded by three conserved regions: the N-terminal WxxD, the central R(R/K)HHCR, and the C-terminal R(V/I)C motifs (Fig. 1a). The basic motif in the (R/K)(R/K)HHCR region forms a β-strand, which creates a positively charged pocket that specifically binds to PI3P ...
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... Image fluorescent proteins in Arabidopsis root cells using a 63Â water-immersion lens (see Note 11). As shown in Fig. 1d, GFP-FREE1 colocalize with the MVB marker mRFP-Rha1. However, wortmannin treatment induces the homotypic fusion and enlargement of MVBs [19]. Under wortmannin treatment, GFP-FREE1 localizes to abnormally large endosomes also labeled by mRFP-Rha1, confirming the MVB localization of GFP-FREE1 in plant cells (see Note ...
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Citations
... Among them, the protein encoded by PuG11 gene contains FYVE domain. The FYVE domain is a zincfinger binding domain that notably occurs in fungi, metazoans, and plant (Gaullier et al., 1998;Shen et al., 2020). Proteins that contain the FYVE zinc-finger domain are recruited to PtdIns3P-containing membranes, participating in numerous biological processes such as membrane trafficking, cytoskeletal regulation, and receptor signaling. ...
Polyporus umbellatus is an edible and medicinal mushroom with the capacity to produce sclerotia. However, the mechanism of P. umbellatus sclerotia formation is unclear. CRZ1 is a C2H2 family transcription factor involved in the Ca2+-calcineurin signaling pathway, which has the function of regulating sclerotia formation, maintaining ion homeostasis, and responding to stress. In this study, we identified 28 C2H2 transcription factors in P. umbellatus genome, 13 of which are differentially expressed between mycelium and sclerotia, including PuCRZ1. Combining DNA affinity purification and sequencing (DAP-seq) and quantitative real-time PCR (qRT-PCR), three genes (PuG10, PuG11, PuG12) were identified as putative PuCRZ1 target genes containing a putative binding motif (GTGGCG) within their promoter. Yeast single hybridization (Y1H) and EMSA further confirmed that PuCRZ1 can bind to the promoter region of PuG10, PuG11, and PuG12. PuCRZ1 gene could reduce the sensitivity of NaCl in yeast cells. Furthermore, overexpression of the PuCRZ1 target gene, especially the FVLY domain containing gene PuG11, could improve the mycelia growth rate and mannitol tolerance in P. umbellatus. These results demonstrate that PuCRZ1 in the Ca2+-calcineurin signaling pathway plays an important role in mycelia growth, as well as osmotic stress tolerance.
... Unexpectedly, the SPI PH domains do not bind phospholipids [106], suggesting that the binding of SPI to endosomal membranes is phospholipid-independent. About fifteen genes encoding FYVE domain-containing proteins are reported in the Arabidopsis genome [107], where thirteen of them do not have homologous sequences in yeast and mammals [108]. Two Arabidopsis FYVE domain-containing PtdIns(3,5)-kinases, FAB1A and FAB1B, play a significant role in the plant cellular environment, and depletion of their expression leads to pollen abortion, delayed endocytosis, acidification, or abnormal vacuole formation [108][109][110]. ...
... About fifteen genes encoding FYVE domain-containing proteins are reported in the Arabidopsis genome [107], where thirteen of them do not have homologous sequences in yeast and mammals [108]. Two Arabidopsis FYVE domain-containing PtdIns(3,5)-kinases, FAB1A and FAB1B, play a significant role in the plant cellular environment, and depletion of their expression leads to pollen abortion, delayed endocytosis, acidification, or abnormal vacuole formation [108][109][110]. The plant FYVE domain protein FREE1 has been reported for cargo membrane protein sorting, vacuole formation, multivesicular body biogenesis, and autophagic-mediated degradation [111][112][113]. ...
Phafin2, a member of the Phafin family of proteins, contributes to a plethora of cellular activities including autophagy, endosomal cargo transportation, and macropinocytosis. The PH and FYVE domains of Phafin2 play key roles in membrane binding, whereas the C-terminal poly aspartic acid (polyD) motif specifically autoinhibits the PH domain binding to the membrane phosphatidylinositol 3-phosphate (PtdIns3P). Since the Phafin2 FYVE domain also binds PtdIns3P, the role of the polyD motif remains unclear. In this study, bioinformatics tools and resources were employed to determine the concurrence of the PH-FYVE module with the polyD motif among Phafin2 and PH-, FYVE-, or polyD-containing proteins from bacteria to humans. FYVE was found to be an ancient domain of Phafin2 and is related to proteins that are present in both prokaryotes and eukaryotes. Interestingly, the polyD motif only evolved in Phafin2 and PH- or both PH-FYVE-containing proteins in animals. PolyD motifs are absent in PH domain-free FYVE-containing proteins, which usually display cellular trafficking or autophagic functions. Moreover, the prediction of the Phafin2-interacting network indicates that Phafin2 primarily cross-talks with proteins involved in autophagy, protein trafficking, and neuronal function. Taken together, the concurrence of the polyD motif with the PH domain may be associated with complex cellular functions that evolved specifically in animals.
