Plant AP180 N-Terminal Homolog Proteins Are Involved in Clathrin-Dependent Endocytosis during Pollen Tube Growth in Arabidopsis thaliana

Abstract

Polarized cell growth in plants is maintained under the strict control and exquisitely choreographed balance of exocytic and endocytic membrane trafficking. The pollen tube has become a model system for rapid polar growth in which delivery of cell wall material and membrane recycling are controlled by membrane trafficking. Endocytosis plays an important role that is poorly understood. The plant AP180 N-Terminal Homolog (ANTH) proteins are putative homologs of Epsin 1 that recruits clathrin to phosphatidylinositol 4, 5-bisphosphate (PIP2) containing membranes to facilitate vesicle budding during endocytosis. Two Arabidopsis ANTH encoded by the genes AtAP180 and AtECA2 are highly expressed in pollen tubes. Pollen tubes from T-DNA inserted knockout mutant lines display significant morphological defects and unique pectin deposition. Fluorescent tagging reveals organization into dynamic foci located at the lateral flanks of the pollen tube. This precisely defined subapical domain coincides which clathrin-mediated endocytosis (CME) and PIP2 localization. Using a liposome-protein binding test, we showed that AtECA2 protein and ANTH domain recombinant proteins have strong affinity to PIP2 and phosphatidic acid containing liposomes in vitro. Taken together these data suggest that Arabidopsis ANTH proteins may play an important role in CME, proper cell wall assembly and morphogenesis.

EPSIN1 Modulates the Plasma Membrane Abundance of FLAGELLIN SENSING2 for Effective Immune Responses

“…These six EPSs form a subfamily that is distinct from the divergent ENTH domain-containing MODIFIED TRANSPORT TO THE VACUOLE, with roles in TGN/ EE-to-vacuolar cargo trafficking (Sauer et al, 2013). The Arabidopsis ENTH subfamily also clusters away from the large ANTH protein family that includes EPSIN-LIKE CLATHRIN ADAPTOR4 (ECA4), PHOSPHATI-DYLINOSITOL BINDING CLATHRIN ASSEMBLY PROTEIN5A (PICALM5; also called ECA2), PICALM5b, and AP180, for which physiological roles have been recently described using loss-of-function mutants (Sauer et al, 2013;Zouhar and Sauer, 2014;Muro et al, 2018;Nguyen et al, 2018;Kaneda et al, 2019). Thus, our identification of EPS1 modulating immune responses expands the short list of Arabidopsis ENTH/ANTH/ VHS domain family members with known physiological functions.…”

Protein Amphipathic Helix Insertion: A Mechanism to Induce Membrane Fission

“…The CALM protein, also known as PICALM, is also involved in fission of CCVs during CME (Miller et al, 2015). CALM contains a N-terminal ANTH domain that is approximately twice as big as the ENTH domain, but has the same core of α-helices (Duncan and Payne, 2003;Legendre-Guillemin et al, 2004;Takatori and Tomita, 2018;Kaneda et al, 2019). ENTH and ANTH domains are so similar that several ANTH domains were originally designated ENTH domains, although later these two domains were subclassified as two distinct domains, ENTH and ANTH (Ford et al, 2002).…”

“…Most ENTH and ANTH domains are lipid-binding domains, they interact specifically with phosphoinositides present in the membrane. However, the mechanisms of the interaction of ENTH and ANTH domains with PtdIns(4,5)P 2 are quite different (Kaneda et al, 2019; and references therein).…”

“…Proteins containing ENTH/ANTH domains (epsins 1, 2, 3 and CALM) are somewhat similar and can be classified as separate subfamily of AH-containing fission-inducing proteins. Possibly, the plant ANTH domain-containing protein AP180 also belongs to this subfamily (Kaneda et al, 2019). Similarly, new fission proteins belonging to this subfamily might be discovered in the future, based on the identification of AHs in their structure.…”

Exocytosis and Endocytosis: Yin-Yang Crosstalk for Sculpting a Dynamic Growing Pollen Tube Tip

“…Therefore, retrograde endocytosis which is the opposite vesicle trafficking in the pollen tube tip, counteracts with anterograde exocytosis to maintain a dynamic balance in the apical dome ( Figure 1 ). It takes place simultaneously to recycle the excessive fusion-unsuccessful exocytic vesicles for multiple rounds of secretion and fusion, and internalize vesicles invaginated from the apical PM ( Wang et al, 2005 ; Zonia and Munnik, 2008 ; Grebnev et al, 2017 ; Kaneda et al, 2019 ). It functions in pollen tube guidance, signal transduction and nutrient uptake ( Sekeres et al, 2015 ; Hao et al, 2016 ; Hempel et al, 2017 ; Wang et al, 2020 ).…”