Characterization of CAP1 and ECA4 adaptors participating in clathrin-mediated endocytosis

Abstract

Formation of endomembrane vesicles is crucial in all eukaryotic cells and relies on vesicle coats such as clathrin. Clathrin-coated vesicles form at the plasma membrane and the trans-Golgi Network. They contain adaptor proteins, which serve as binding bridges between clathrin, vesicle membranes, and cargoes. A large family of monomeric ANTH/ENTH/VHS adaptors is present in A. thaliana. Here, we characterize two homologous ANTH-type clathrin adaptors, CAP1 and ECA4, in clathrin-mediated endocytosis (CME). CAP1 and ECA4 are recruited to sites at the PM identified as clathrin-coated pits (CCPs), where they occasionally exhibit early bursts of high recruitment. Subcellular binding preferences of N- and C-terminal fluorescent protein fusions of CAP1 identified a functional adaptin-binding motif in the unstructured tails of CAP1 and ECA4. In turn, no function can be ascribed to a double serine phosphorylation site conserved in these proteins. Double knockout mutants do not exhibit deficiencies in general development or CME, but a contribution of CAP1 and ECA4 to these processes is revealed in crosses into sensitized endocytic mutant backgrounds. Overall, our study documents a contribution of CAP1 and ECA4 to CME in A. thaliana and opens questions about functional redundancy among non-homologous vesicle coat components.

Membrane trafficking functions of the ANTH/ENTH/VHS domain‐containing proteins in plants

“…PICALM4b/CAP1 has also been reported to interact with clathrin light chain1 (CLC1) and the putative clathrin‐uncoating factors auxilin‐like1 and 2 [48]. Although a double mutation in the paralogous PICALM4a / ECA4 and PICALM4b / CAP1 does not significantly affect their endocytic activities, this double mutation affects clathrin‐mediated endocytosis with mutations in other endocytosis‐related genes, such as TASH and SH3P s [49]. TASH and SH3P s are genes that encode for a subunit of the TPC, and BAR‐ and SH3 domain‐containing proteins, respectively.…”