Cargo- and adaptor-specific mechanisms regulate clathrin-mediated endocytosis

Clathrin-mediated endocytosis of surface receptors and their bound ligands (i.e., cargo) is highly regulated, including by the cargo itself. One of the possible sources of the observed heterogeneous dynamics of clathrin-coated pits (CCPs) might be the different cargo content. Consistent with this, we show that CCP size and dynamic behavior varies with low density lipoprotein receptor (LDLR) expression levels in a manner dependent on the LDLR-specific adaptors, Dab2 and ARH. In Dab2-mCherry–expressing cells, varying LDLR expression leads to a progressive increase in CCP size and to the appearance of nonterminal endocytic events. In LDLR and ARH-mCherry–expressing cells in addition to an increase in CCP size, turnover of abortive CCPs increases, and the rate of CCP maturation decreases. Altogether, our results underscore the highly dynamic and cargo-responsive nature of CCP assembly and suggest that the observed heterogeneity is, in part, related to compositional differences (e.g., cargo and adaptors) between CCPs.     

Arabidopsis ROP9 and ROP10 GTPases differentially regulate auxin and ABA responses

Auxin and abscisic acid (ABA) are major plant hormones that act together to modulate numerous aspects of plant growth and development, including seed germination, primary root elongation, and lateral root formation. In this study, we analyzed the loss-of-function mutants of two closely related ROP (Rho of plants) GTPases, ROP9 and ROP10, and found that these ROP GTPases differentially regulate the auxin and ABA responses. rop9 and rop10 mutations enhanced the ABA-induced suppression of seed germination, primary root growth, and lateral root formation and the expression of ABA-responsive genes, whereas rop9 but not rop10 suppressed auxin-induced root phenotypes and auxin-responsive gene expression. These results suggest that both ROP9 and ROP10 function as negative regulators of ABA signaling, and that ROP9, but not ROP10, functions as a positive regulator of auxin signaling. Previously, ROPinteractive CRIB motif-containing protein 1 (RIC1) was reported to participate in auxin and ABA responses, and to have a similar effect as ROP9 and ROP10 on gene expression, root development, and seed germination. Because RIC proteins mediate ROP GTPase signaling, our results suggest that ROP9 and ROP10 GTPases function upstream of RIC1 in auxin- and ABA-regulated root development and seed germination.     

A ROP GTPase-dependent auxin signaling pathway regulates the subcellular distribution of PIN2 in Arabidopsis roots

PIN-FORMED (PIN) protein-mediated auxin polar transport is critically important for development, pattern formation, and morphogenesis in plants. Auxin has been implicated in the regulation of polar auxin transport by inhibiting PIN endocytosis, but how auxin regulates this process is poorly understood. Our genetic screen identified the Arabidopsis SPIKE1 (SPK1) gene whose loss-of-function mutations increased lateral root density and retarded gravitropic responses, as do pin2 knockout mutations. SPK1 belongs to the conserved DHR2-Dock family of Rho guanine nucleotide exchange factors. The spk1 mutations induced PIN2 internalization that was not suppressed by auxin, as did the loss-of-function mutations for Rho-like GTPase from Plants 6 (ROP6)-GTPase or its effector RIC1. Furthermore, SPK1 was required for auxin induction of ROP6 activation. Our results have established a Rho GTPase-based auxin signaling pathway that maintains PIN2 polar distribution to the plasma membrane via inhibition of its internalization in Arabidopsis roots. Our findings provide new insights into signaling mechanisms that underlie the regulation of the dynamic trafficking of PINs required for long-distance auxin transport and that link auxin signaling to PIN-mediated pattern formation and morphogenesis.     

Multiple mechanisms collectively regulate clathrin-mediated endocytosis of the epidermal growth factor receptor

Endocytosis of the epidermal growth factor receptor (EGFR) is important for the regulation of EGFR signaling. However, EGFR endocytosis mechanisms are poorly understood, which precludes development of approaches to specifically inhibit EGFR endocytosis and analyze its impact on signaling. Using a combination of receptor mutagenesis and RNA interference, we demonstrate that clathrin-dependent internalization of activated EGFR is regulated by four mechanisms, which function in a redundant and cooperative fashion. These mechanisms involve ubiquitination of the receptor kinase domain, the clathrin adaptor complex AP-2, the Grb2 adaptor protein, and three C-terminal lysine residues (K1155, K1158, and K1164), which are acetylated, a novel posttranslational modification for the EGFR. Based on these findings, the first internalization-defective EGFR mutant with functional kinase and normal tyrosine phosphorylation was generated. Analysis of the signaling kinetics of this mutant revealed that EGFR internalization is required for the sustained activation of protein kinase B/AKT but not for the activation of mitogen-activated protein kinase.     

ROPGEF1 and ROPGEF4 are functional regulators of ROP11 GTPase in ABA-mediated stomatal closure in Arabidopsis

ROPs constitute a family of plant-specific, RHO-like small GTPases that serve as molecular switches in a wide range of signaling pathways. The activities of ROPs are regulated by guanine nucleotide exchange factors (GEFs). ROP11, a member of the ROP GTPase family in Arabidopsis, is a negative regulator of multiple ABA responses. In this study, we show that ROPGEF1 and ROPGEF4 interact with ROP11 on plasma membranes in guard cells. Furthermore, our analyses of ROPGEF1/4 knockout mutants and overexpressing lines suggested that ROPGEF1 and ROPGEF4 are specific regulators of ROP11 function in ABA-mediated stomatal closure.     

Dynamin GTPase domain mutants that differentially affect GTP binding, GTP hydrolysis, and clathrin-mediated endocytosis

The GTPase dynamin is essential for clathrin-mediated endocytosis. Unlike most GTPases, dynamin has a low affinity for nucleotide, a high rate of GTP hydrolysis, and can self-assemble, forming higher order structures such as rings and spirals that exhibit up to 100-fold stimulated GTPase activity. The role(s) of GTP binding and/or hydrolysis in endocytosis remain unclear because mutations in the GTPase domain so far studied impair both. We generated a new series of GTPase domain mutants to probe the mechanism of GTP hydrolysis and to further test the role of GTP binding and/or hydrolysis in endocytosis. Each of the mutations had parallel effects on assembly-stimulated and basal GTPase activities. In contrast to previous reports, we find that mutation of Thr-65 to Ala (or Asp or His) dramatically lowered both the rate of assembly-stimulated GTP hydrolysis and the affinity for GTP. The assemblystimulated rate of hydrolysis was lowered by the mutation of Ser-61 to Asp and increased by the mutation of Thr-141 to Ala without significantly altering the Km for GTP. For some mutants and to a lesser extent for WT dynamin, self-assembly dramatically altered the Km for GTP, suggesting that conformational changes in the active site accompany self-assembly. Analysis of transferrin endocytosis rates in cells overexpressing mutant dynamins revealed a stronger correlation with both the basal and assembly-stimulated rates of GTP hydrolysis than with the calculated ratio of dynamin-GTP/free dynamin, suggesting that GTP binding is not sufficient, and GTP hydrolysis is required for clathrin-mediated endocytosis in vivo.     

Diverse nitrogen signals activate convergent ROP2-TOR signaling in Arabidopsis

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Accessory protein recruitment motifs in clathrin-mediated endocytosis

Clathrin-mediated endocytosis depends upon the interaction of accessory proteins with the alpha-ear of the AP-2 adaptor. We present structural characterization of these regulatory interactions. DPF and DPW motif peptides derived from eps15 and epsin bind in type I beta turn conformations to a conserved pocket on the alpha-ear platform. We show evidence for a second binding site that is DPW motif specific. The structure of a complex with an AP-2 binding segment from amphiphysin reveals a novel binding motif that we term FxDxF, which is engaged in an extended conformation by a unique surface of the platform domain. The FxDxF motif is also used by AP180 and the 170 kDa isoform of synaptojanin and can be found in several potential endocytic proteins, including HIP1, CD2AP, and PLAP. A mechanism of clathrin assembly regulation is suggested by three different AP-2 engagement modes.     

Myosin 1E coordinates actin assembly and cargo trafficking during clathrin-mediated endocytosis

Myosin 1E (Myo1E) is recruited to sites of clathrin-mediated endocytosis coincident with a burst of actin assembly. The recruitment dynamics and lifetime of Myo1E are similar to those of tagged actin polymerization regulatory proteins. Like inhibition of actin assembly, depletion of Myo1E causes reduced transferrin endocytosis and a significant delay in transferrin trafficking to perinuclear compartments, demonstrating an integral role for Myo1E in these actin-mediated steps. Mistargeting of GFP-Myo1E or its src-homology 3 domain to mitochondria results in appearance of WIP, WIRE, N-WASP, and actin filaments at the mitochondria, providing evidence for Myo1E's role in actin assembly regulation. These results suggest for mammalian cells, similar to budding yeast, interdependence in the recruitment of type I myosins, WIP/WIRE, and N-WASP to endocytic sites for Arp2/3 complex activation to assemble F-actin as endocytic vesicles are being formed.     

ROP/RAC GTPase: an old new master regulator for plant signaling

The ROP family of small GTPases has emerged as a versatile and pivotal regulator in plant signal transduction. Recent studies have implicated ROP signaling in diverse processes ranging from cytoskeletal organization to hormone and stress responses. Acting as a switch early in signaling cascades, ROPs are also capable of orchestrating several downstream pathways to amplify a specific signal.     

EphA8-ephrinA5 signaling and clathrin-mediated endocytosis is regulated by Tiam-1, a Rac-specific guanine nucleotide exchange factor

Recent studies indicate that endocytosis of Eph-ephrin complexes may be one of the mechanisms by which a high affinity cell-cell adhesion is converted to a repulsive interaction. In this study, we show that EphA8 undergoes clathrin-mediated endocytosis upon treatment with ephrin-A5, and that EphA8 is associated tightly with Tiam-1, a Rac-specific guanine nucleotide exchange factor. Analysis of EphA8 deletion mutants revealed that a juxtamembrane region in EphA8 is critically involved in endocytosis of EphA8-ephrinA5 complexes. An EphA8 mutant lacking this juxtamembrane portion was defective for endocytosis with ephrinA5, and also displayed a weak association with Tiam-1. Expression of an endocytosis-defective version of EphA8 resulted in a low level of Rac activity in response to ephrin-A5 stimulation. More importantly, down-regulation of Tiam-1 resulted in inefficient endocytosis of EphA8-ephrinA5 complexes. These results suggest that Tiam-1 plays a role in clathrin-dependent endocytosis of EphA8-ephrinA5 complexes.     

Differential requirements for AP-2 in clathrin-mediated endocytosis

AP-2 complexes are key components in clathrin-mediated endocytosis (CME). They trigger clathrin assembly, interact directly with cargo molecules, and recruit a number of endocytic accessory factors. Adaptor-associated kinase (AAK1), an AP-2 binding partner, modulates AP-2 function by phosphorylating its mu2 subunit. Here, we examined the effects of adenoviral-mediated overexpression of WT AAK1, kinase-dead, and truncation mutants in HeLa cells, and show that AAK1 also regulates AP-2 function in vivo. WT AAK1 overexpression selectively blocks transferrin (Tfn) receptor and LRP endocytosis. Inhibition was kinase independent, but required the full-length AAK1 as truncation mutants were not inhibitory. Although changes in mu2 phosphorylation were not detected, AAK1 overexpression significantly decreased the phosphorylation of large adaptin subunits and the normally punctate AP-2 distribution was dispersed, suggesting that AAK1 overexpression inhibited Tfn endocytosis by functionally sequestering AP-2. Surprisingly, clathrin distribution and EGF uptake were unaffected by AAK1 overexpression. Thus, AP-2 may not be stoichiometrically required for coat assembly, and may have a more cargo-selective function in CME than previously thought.     

Molecular mechanism and physiological functions of clathrin-mediated endocytosis

Clathrin-mediated endocytosis is the endocytic portal into cells through which cargo is packaged into vesicles with the aid of a clathrin coat. It is fundamental to neurotransmission, signal transduction and the regulation of many plasma membrane activities and is thus essential to higher eukaryotic life. Morphological stages of vesicle formation are mirrored by progression through various protein modules (complexes). The process involves the formation of a putative FCH domain only (FCHO) initiation complex, which matures through adaptor protein 2 (AP2)-dependent cargo selection, and subsequent coat building, dynamin-mediated scission and finally auxilin- and heat shock cognate 70 (HSC70)-dependent uncoating. Some modules can be used in other pathways, and additions or substitutions confer cell specificity and adaptability.     

Functional partnership between amphiphysin and dynamin in clathrin-mediated endocytosis

Amphiphysin, a protein that is highly concentrated in nerve terminals, has been proposed to function as a linker between the clathrin coat and dynamin in the endocytosis of synaptic vesicles. Here, using a cell-free system, we provide direct morphological evidence in support of this hypothesis. Unexpectedly, we also find that amphiphysin-1, like dynamin-1, can transform spherical liposomes into narrow tubules. Moreover, amphiphysin-1 assembles with dynamin-1 into ring-like structures around the tubules and enhances the liposome-fragmenting activity of dynamin-1 in the presence of GTP. These results show that amphiphysin binds lipid bilayers, indicate a potential function for amphiphysin in the changes in bilayer curvature that accompany vesicle budding, and imply a close functional partnership between amphiphysin and dynamin in endocytosis.     

Isoprenylcysteine methylation and demethylation regulate abscisic acid signaling in Arabidopsis

Isoprenylated proteins bear an isoprenylcysteine methyl ester at the C terminus. Although isoprenylated proteins have been implicated in meristem development and negative regulation of abscisic acid (ABA) signaling, the functional role of the terminal methyl group has not been described. Here, we show that transgenic Arabidopsis thaliana plants overproducing isoprenylcysteine methyltransferase (ICMT) exhibit ABA insensitivity in stomatal closure and seed germination assays, establishing ICMT as a negative regulator of ABA signaling. By contrast, transgenic plants overproducing isoprenylcysteine methylesterase (ICME) exhibit ABA hypersensitivity in stomatal closure and seed germination assays. Thus, ICME is a positive regulator of ABA signaling. To test the hypothesis that ABA signaling is under feedback regulation at the level of isoprenylcysteine methylation, we examined the effect of ABA on ICMT and ICME gene expression. Interestingly, ABA induces ICME gene expression, establishing a positive feedback loop whereby ABA promotes ABA responsiveness of plant cells via induction of ICME expression, which presumably results in the demethylation and inactivation of isoprenylated negative regulators of ABA signaling. These results suggest strategies for metabolic engineering of crop species for drought tolerance by targeted alterations in isoprenylcysteine methylation.