A Second Las17 Monomeric Actin‐Binding Motif Functions in Arp2/3‐Dependent Actin Polymerization During Endocytosis

Abstract

During clathrin-mediated endocytosis (CME), actin assembly provides force to drive vesicle internalization. Members of the Wiskott–Aldrich syndrome protein (WASP) family play a fundamental role stimulating actin assembly. WASP family proteins contain a WH2 motif that binds globular actin (G-actin) and a central-acidic motif that binds the Arp2/3 complex, thus promoting the formation of branched actin filaments. Yeast WASP (Las17) is the strongest of five factors promoting Arp2/3-dependent actin polymerization during CME. It was suggested that this strong activity may be caused by a putative second G-actin-binding motif in Las17. Here, we describe the in vitro and in vivo characterization of such Las17 G-actin-binding motif (LGM) and its dependence on a group of conserved arginine residues. Using the yeast two-hybrid system, GST-pulldown, fluorescence polarization and pyrene-actin polymerization assays, we show that LGM binds G-actin and is necessary for normal Arp2/3-mediated actin polymerization in vitro. Live-cell fluorescence microscopy experiments demonstrate that LGM is required for normal dynamics of actin polymerization during CME. Further, LGM is necessary for normal dynamics of endocytic machinery components that are recruited at early, intermediate and late stages of endocytosis, as well as for optimal endocytosis of native CME cargo. Both in vitro and in vivo experiments show that LGM has relatively lower potency compared to the previously known Las17 G-actin-binding motif, WH2. These results establish a second G-actin-binding motif in Las17 and advance our knowledge on the mechanism of actin assembly during CME.

New Regulators of Clathrin-Mediated Endocytosis Identified in Saccharomyces cerevisiae by Systematic Quantitative Fluorescence Microscopy

“…To test that possibility, MATa strains expressing the 319 GFP-tagged proteins in these categories were mated with MATa cells expressing Sla1-RFP from the endogenous locus and resulting diploid cells were selected with appropriate markers. Sla1 is a multifunctional clathrin adaptor and actin polymerization regulator present at all sites of CME and easily visible by fluorescence microscopy ( Figure 1A) (Ayscough et al 1999;Kaksonen et al 2003;Kaksonen et al 2005;Di Pietro et al 2010;Feliciano and Di Pietro 2012;Feliciano et al 2015). Each diploid strain expressing both the corresponding GFP-fusion protein and Sla1-RFP was subjected to live cell confocal fluorescence microscopy and colocalization analysis.…”

“…GST-and polyhistidine-fusion proteins were expressed in Escherichea coli and purified as described (Feliciano et al 2011(Feliciano et al , 2015. GST-pulldowns were performed by loading glutathioneSepharose beads with GST-fusion protein (5 mg) for 30 min at room temperature.…”

Novel function of a dynein light chain in actin assembly during clathrin-mediated endocytosis

“…GST-pulldown assays were performed as previously described ( Farrell et al, 2015 ; Feliciano et al, 2015 ). In brief, recombinant polyhistidine-tagged proteins (10 µg) were incubated with GST and GST fusion proteins (10 µg) bound to glutathione-Sepharose beads in 1 ml of PBS containing 0.1% Triton X-100 for 1 h at 4°C.…”

WASP family proteins, more than Arp2/3 activators

“…More recently, work from Feliciano et al [23] demonstrated the importance of arginine residue pairs (RR349,350; RR382,383) in Las17 that facilitated G-actin binding within the PPR region. Their study focussed on the role of these residues in the context of Arp2/3 and clearly highlighted the importance of the RR pairs for G-actin binding and for rapid polymerization by Arp2/3.…”