Abstract
Clathrin-mediated endocytosis (CME) is the best-characterized type of endocytosis in eukaryotic cells. Plants appear to possess all of the molecular components necessary to carry out CME; however, functional characterization of the components is still in its infancy. A yeast two-hybrid screen identified μ2 as a putative interaction partner of CELLULOSE SYNTHASE6 (CESA6). Arabidopsis (Arabidopsis thaliana) μ2 is homologous to the medium subunit 2 of the mammalian ADAPTOR PROTEIN COMPLEX2 (AP2). In mammals, the AP2 complex acts as the central hub of CME by docking to the plasma membrane while concomitantly recruiting cargo proteins, clathrin triskelia, and accessory proteins to the sites of endocytosis. We confirmed that μ2 interacts with multiple CESA proteins through the μ-homology domain of μ2, which is involved in specific interactions with endocytic cargo proteins in mammals. Consistent with its role in mediating the endocytosis of cargos at the plasma membrane, μ2-YELLOW FLUORESCENT PROTEIN localized to transient foci at the plasma membrane, and loss of μ2 resulted in defects in bulk endocytosis. Furthermore, loss of μ2 led to increased accumulation of YELLOW FLUORESCENT PROTEIN-CESA6 particles at the plasma membrane. Our results suggest that CESA represents a new class of CME cargo proteins and that plant cells might regulate cellulose synthesis by controlling the abundance of active CESA complexes at the plasma membrane through CME.
Highlights
Clathrin-mediated endocytosis (CME) is the best-characterized type of endocytosis in eukaryotic cells
Genetic and coimmunoprecipitation studies have indicated that CELLULOSE SYNTHASE1 (CESA1), CESA3, and CELLULOSE SYNTHASE6 (CESA6)-like (CESA6, CESA2, CESA5, and CESA9) isoforms are constituents of cellulose synthase complexes (CSCs) during primary cell wall synthesis (Persson et al, 2005; Desprez et al, 2007; Persson et al, 2007; Wang et al, 2008), whereas CESA4, CESA7, and CESA8 are implicated in the cellulose synthesis of secondary cell walls (Taylor et al, 1999, 2003; Brown et al, 2005)
The function of many CME proteins has been extensively characterized in mammals (McMahon and Boucrot, 2011), and homologs of many CME components are encoded by the Arabidopsis genome, including multiple copies of clathrin H chain and clathrin light chain (CLC), all four subunits of the heterotetrameric ADAPTOR PROTEIN COMPLEX2 (AP2) complex, dynamin-related proteins, and accessory proteins such as AP180 (Holstein, 2002; Chen et al, 2011); many CME components have yet to be characterized in plants
Summary
M2 Directly Interacts with CESA Proteins m2 was identified as a putative interaction partner of the central domain of CESA6 in a conventional Y2H screen (Supplemental Fig. S1). The localization and temporal behavior of m2-YFP and CLC-mOrange in dark-grown hypocotyls are similar to those of CLC and dynamin-related proteins (DRP1A and DRP2B) in other cell types that have been observed in previous studies (Konopka et al, 2008; Konopka and Bednarek, 2008; Fujimoto et al, 2010), which provides support for m2 playing a role in CME. To investigate whether the increased density of CESAs at the plasma membrane in m2-1 influenced the behavior of CESAs, the motility of plasma membranelocalized YFP-CESA6 particles was measured in both the m2-1 prc and prc genetic backgrounds In both lines, YFP-CESA6 particles traveled bidirectionally at constant velocities along linear tracks that can be visualized in averaged projections of 5-min time-lapse images (Supplemental Fig. S5A). This observation suggests that the distribution of CESAs is affected by m2-1, the function of YFP-CESA6 at the plasma membrane may not be significantly affected by m2-1
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