Caenorhabditis elegans auxilin: a J-domain protein essential for clathrin-mediated endocytosis in vivo

Greener, Tsvika; Grant, Barth D.; Zhang, Yinhua; Wu, Xufeng; Greene, Lois E.; Hirsh, David; Eisenberg, Evan

doi:10.1038/35055137

Cited by 92 publications

(112 citation statements)

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“…12, July 2001function similar to mammalian auxilin (Greener et al, 2001). We also identified a repeated sequence in RME-8 that is present in four copies.…”

Section: Elegans Rme-8 In Endosomal Traffickingmentioning

confidence: 68%

RME-8, a Conserved J-Domain Protein, Is Required for Endocytosis inCaenorhabditis elegans

Zhang

Grant

Hirsh

2001

MBoC

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154

202

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By genetic analysis of Caenorhabditis elegans mutants defective in yolk uptake, we have identified new molecules functioning in the endocytosis pathway. Here we describe a novel J-domaincontaining protein, RME-8, identified by such genetic analysis. RME-8 is required for receptormediated endocytosis and fluid-phase endocytosis in various cell types and is essential for C. elegans development and viability. In the macrophage-like coelomocytes, RME-8 localizes to the limiting membrane of large endosomes. Endocytosis markers taken up by the coelomocytes rapidly accumulate in these large RME-8 -positive endosomes, concentrate in internal subendosomal structures, and later appear in RME-8 -negative lysosomes. rme-8 mutant coelomocytes fail to accumulate visible quantities of endocytosis markers. These observations show that RME-8 functions in endosomal trafficking before the lysosome. RME-8 homologues are found in multicellular organisms from plants to humans but not in the yeast Saccharomyces cerevisiae. These sequence homologies suggest that RME-8 fulfills a conserved function in multicellular organisms. INTRODUCTIONIntracellular trafficking of endocytosed molecules requires an elaborate network of organelles (Mellman, 1996;Mukherjee et al., 1997). The compartments within the endocytic network were established in mammalian cells by studying the trafficking kinetics of ligands and receptors (such as transferrin and transferrin receptor, epidermal growth factor [EGF], and EGF-receptor, and low-density lipoprotein [LDL] and LDL receptor) and of fluid-phase markers. Ligands and receptors are often internalized through clathrin-coated pits and vesicles and then are targeted to early or sorting endosomes by vesicle fusion. In the sorting endosome, many ligands can dissociate from their receptors because of a low pH environment. The ligands bound for degradation are then sorted and targeted to the late endosome (Dunn and Maxfield, 1992). Recycling receptors are sorted to an endocytic recycling compartment, from which they recycle back to the plasma membrane (van der Sluijs et al., 1992). In the late endosome, ligands and other lysosome targeted molecules are further sorted, concentrated, and delivered to the lysosome for enzymatic degradation (Futter et al., 1996).Each of these trafficking steps occurs in a specific vesicular compartment. Despite the continuous flow of endocytosed molecules, these compartments are characterized by unique morphologies, sizes, intravesicular pH levels, membrane proteins, and lipid compositions, although these characteristics can vary with different cell types (Mellman, 1996;Mukherjee et al., 1997;Odorizzi et al., 2000). In mammalian cells, sorting endosomes have a tubular-vesicular morphology with an internal pH ϳ6.0 (Mukherjee et al., 1997). Late endosomes are similar in size to sorting endosomes but have characteristic internal vesicles formed by the invagination of late endosomal membranes (Hirsch et al., 1968;Futter et al., 1996). Two alternative models were proposed to describe the t...

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“…12, July 2001function similar to mammalian auxilin (Greener et al, 2001). We also identified a repeated sequence in RME-8 that is present in four copies.…”

Section: Elegans Rme-8 In Endosomal Traffickingmentioning

confidence: 68%

RME-8, a Conserved J-Domain Protein, Is Required for Endocytosis inCaenorhabditis elegans

Zhang

Grant

Hirsh

2001

MBoC

Self Cite

154

202

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“…For N-terminally His 6 -tagged Ydj1p, the YDJ1 gene was amplified and cloned into the NdeI and BamHI sites of pET15b (pET15b-YDJ1; a gift from S. Burston). For C-terminally His 6 -tagged Swa2p, the PCR-amplified SWA2 gene was cloned into pET21a (Novagen) using NdeI and BamHI restriction sites (pET21a-SWA2).…”

Section: Methodsmentioning

confidence: 99%

Clathrin Coat Disassembly by the Yeast Hsc70/Ssa1p and Auxilin/Swa2p Proteins Observed by Single-particle Burst Analysis Spectroscopy

Krantz

Puchalla

Thapa

et al. 2013

Journal of Biological Chemistry

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“…Convincing demonstration of its in vivo function followed identification of the protein auxilin as its specific cochaperone (Ungewickell et al, 1995). Inactivation of yeast auxilin (Swa2) leads to anomalous accumulation of coated vesicles (Gall et al, 2000;Pishvaee et al, 2000), and RNA interference with auxilin in nematodes causes similar defects (Greener et al, 2001). Mammalian cells express a brain-specific auxilin1 and a ubiquitous auxilin2 (also called cyclin G-associated kinase; GAK) Umeda et al, 2000).…”

Section: Introductionmentioning

confidence: 99%

A Motif in the Clathrin Heavy Chain Required for the Hsc70/Auxilin Uncoating Reaction

Rapoport

Boll

et al. 2008

MBoC

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The 70-kDa heat-shock cognate protein (Hsc70) chaperone is an ATP-dependent "disassembly enzyme" for many subcellular structures, including clathrin-coated vesicles where it functions as an uncoating ATPase. Hsc70, and its cochaperone auxilin together catalyze coat disassembly. Like other members of the Hsp70 chaperone family, it is thought that ATP-bound Hsc70 recognizes the clathrin triskelion through an unfolded exposed hydrophobic segment. The best candidate is the unstructured C terminus (residues 1631-1675) of the heavy chain at the foot of the tripod below the hub, containing the sequence motif QLMLT, closely related to the sequence bound preferentially by the substrate groove of Hsc70 (Fotin et al., 2004b). To test this hypothesis, we generated in insect cells recombinant mammalian triskelions that in vitro form clathrin cages and clathrin/AP-2 coats exactly like those assembled from native clathrin. We show that coats assembled from recombinant clathrin are good substrates for ATP-and auxilin-dependent, Hsc70-catalyzed uncoating. Finally, we show that this uncoating reaction proceeds normally when the coats contain recombinant heavy chains truncated C-terminal to the QLMLT motif, but very inefficiently when the motif is absent. Thus, the QLMLT motif is required for Hsc-70 -facilitated uncoating, consistent with the proposal that this sequence is a specific target of the chaperone. INTRODUCTIONClathrin-coated vesicles, the best known carrier of intracellular membrane traffic, transport proteins and lipids from the plasma membrane to endosomes and between endosomes and the trans-Golgi network. Polymerization of the principal coat protein clathrin into a lattice-like assembly by sequential addition of individual, cytosolic clathrin trimers to a growing shell shapes the budding coated pit (Ehrlich et al., 2004; for review, see Kirchhausen, 2000). Intermediary proteins, known as adaptors, form the interface between the outer clathrin coat and the incorporated membrane bilayer. These adaptors selectively recruit membrane-anchored proteins ("cargo"). The most prominent adaptors are the heterotetrameric AP-2 and its cousins AP-1, AP-3, and AP-4, but there are other, presumably more specialized, adaptors, such as ␤-arrestins, epsin, GGAs, and dishevelled (Yu et al., 2007; for review, see Owen et al., 2004;Robinson, 2004).Coats can assemble in vitro without incorporated membrane, either from clathrin alone ("cages", stable only at reduced pH) or from clathrin plus heteroteterameric APs ("coats", stable at neutral pH) (Keen et al., 1979;Kirchhausen and Harrison, 1981; Vigers et al., 1986a,b;Kirchhausen, 2000;Fotin et al., 2004b). Coat assembly in vitro can proceed to completion without intervention of other factors. In vivo, coat assembly occurs only on membrane surfaces, and fission (pinching off) of the incorporating bilayer requires the large GTPase, dynamin (Sever, 2002;Praefcke and McMahon, 2004;Kruchten and McNiven, 2006;Macia et al., 2006). Removal of the coat (uncoating), essential for subsequent vesicle f...

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Caenorhabditis elegans auxilin: a J-domain protein essential for clathrin-mediated endocytosis in vivo

Cited by 92 publications

References 26 publications

RME-8, a Conserved J-Domain Protein, Is Required for Endocytosis inCaenorhabditis elegans

RME-8, a Conserved J-Domain Protein, Is Required for Endocytosis inCaenorhabditis elegans

Clathrin Coat Disassembly by the Yeast Hsc70/Ssa1p and Auxilin/Swa2p Proteins Observed by Single-particle Burst Analysis Spectroscopy

A Motif in the Clathrin Heavy Chain Required for the Hsc70/Auxilin Uncoating Reaction

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