Structure of the Functional Fragment of Auxilin Required for Catalytic Uncoating of Clathrin-Coated Vesicles

Gruschus, James M.; Han, Chae J.; Greener, Tsvika; Ferretti, James A.; Greene, Lois E.; Eisenberg, Evan

doi:10.1021/bi0354740

Cited by 29 publications

(33 citation statements)

References 43 publications

(90 reference statements)

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“…The low levels of hdj1 immobilization (in combination with hsp70 HN ) eliminated hdj1/N TAIL interactions that could otherwise confound the analysis of hsp70 HN /N TAIL binding reactions. The relatively low amount of hsp40 hdj1 used is consistent with the ability of hsp40 co-chaperones to function in substoichimetric (catalytic) amounts (Gruschus et al, 2004) and also mimics the small amounts of contaminating bacterial co-chaperon in the commercially available hsp70 AD sample (see Figure 2). Hsp40-hsp70 interactions on the CM5 sensors are possible at these immobilization levels due to the fact that these ligands are covalently linked to long and flexible dextran arms.…”

Section: Hsp40 Enhances N Tail Binding To Hsp70 Based Upon Spr Studiessupporting

confidence: 58%

High affinity binding between Hsp70 and the C‐terminal domain of the measles virus nucleoprotein requires an Hsp40 co‐chaperone

Couturier

Buccellato

Costanzo

et al. 2009

J of Molecular Recognition

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The major inducible 70 kDa heat shock protein (hsp70) binds the measles virus (MeV) nucleocapsid with high affinity in an ATP-dependent manner, stimulating viral transcription and genome replication, and profoundly influencing virulence in mouse models of brain infection. Binding is mediated by two hydrophobic motifs (Box-2 and Box-3) located within the C-terminal domain (N(TAIL)) of the nucleocapsid protein, with N(TAIL) being an intrinsically disordered domain. The current work showed that high affinity hsp70 binding to N(TAIL) requires an hsp40 co-chaperone that interacts primarily with the hsp70 nucleotide binding domain (NBD) and displays no significant affinity for N(TAIL). Hsp40 directly enhanced hsp70 ATPase activity in an N(TAIL)-dependent manner, and formation of hsp40-hsp70-N(TAIL) intracellular complexes required the presence of N(TAIL) Box-2 and 3. Results are consistent with the functional interplay between hsp70 nucleotide and substrate binding domains (SBD), where ATP hydrolysis is rate limiting to high affinity binding to client proteins and is enhanced by hsp40. As such, hsp40 is an essential variable in understanding the outcome of MeV-hsp70 interactions.

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Section: Hsp40 Enhances N Tail Binding To Hsp70 Based Upon Spr Studiessupporting

confidence: 58%

High affinity binding between Hsp70 and the C‐terminal domain of the measles virus nucleoprotein requires an Hsp40 co‐chaperone

Couturier

Buccellato

Costanzo

et al. 2009

J of Molecular Recognition

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“…The clathrin-binding region seems to be largely unstructured on the isolated fragment in solution, but ϳ25 residues at its C-terminal end contribute two additional helices to the usual three of the globular J-domain (Ungewickell et al, 1997;Fotin et al, 2004a;Gruschus et al, 2004). The clathrin trimer is a spider-like "triskelion," which assembles by forming an elaborately interdigitated network ( Figure 1A) (Kirchhausen and Harrison, 1981;Ungewickell and Branton, 1981;Smith et al, 1998;Musacchio et al, 1999;Fotin et al, 2004b).…”

Section: Introductionmentioning

confidence: 99%

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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“…Type III JDPs contain only the conserved J-domain that can be located anywhere in the protein sequence. To this group belong functionally diverse proteins such as E. coli DjlA, the clathrin-uncoating auxilin (11), mitochondrial Tim14 and Tim16 (12), and endoplasmic reticulum Sec63p (13).…”

mentioning

confidence: 99%

Role of DnaJ G/F-rich Domain in Conformational Recognition and Binding of Protein Substrates*

Perales-Calvo¹,

Muga

Moro

2010

Journal of Biological Chemistry

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Structure of the Functional Fragment of Auxilin Required for Catalytic Uncoating of Clathrin-Coated Vesicles

Cited by 29 publications

References 43 publications

High affinity binding between Hsp70 and the C‐terminal domain of the measles virus nucleoprotein requires an Hsp40 co‐chaperone

High affinity binding between Hsp70 and the C‐terminal domain of the measles virus nucleoprotein requires an Hsp40 co‐chaperone

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

Role of DnaJ G/F-rich Domain in Conformational Recognition and Binding of Protein Substrates*

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