Cysteine String Protein Monitors Late Steps in Cystic Fibrosis Transmembrane Conductance Regulator Biogenesis

Zhang, Hui; Schmidt, Béla Z.; Sun, Fei; Condliffe, Steven B.; Butterworth, Michael; Youker, Robert T.; Brodsky, Jeffrey L.; Aridor, Meir; Frizzell, Raymond A.

doi:10.1074/jbc.m512013200

Cited by 45 publications

(42 citation statements)

References 61 publications

(69 reference statements)

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“…Consistent also with a direct chaperone action, both CspWT and CspH43Q prevented the in vitro aggregation of the first nucleotide binding domain 1 (NBD1) of CFTR with the same efficiency (23), and similar data were provided for Hdj-2, another Hsp40 homologue, and its HPD mutant (36). In this study, we show that the overexpression of CspH43Q led to an increase in the level of immature CFTR (band B), exceeding that obtained with Sar1 mutants alone.…”

Section: Discussionsupporting

confidence: 80%

“…Direct Chaperone Function of Csp-Earlier, we showed that Csp binds directly to the R-domain and the N terminus of CFTR (23). Consistent also with a direct chaperone action, both CspWT and CspH43Q prevented the in vitro aggregation of the first nucleotide binding domain 1 (NBD1) of CFTR with the same efficiency (23), and similar data were provided for Hdj-2, another Hsp40 homologue, and its HPD mutant (36).…”

Section: Discussionsupporting

confidence: 64%

“…Csp variants bearing a mutation in their HPD motif are defective in stimulating the ATPase activity of and in binding to Hsc70 (14,23,29). Therefore, it was not surprising that we also found that the amount of Hsc70/Hsp70 co-immunoprecipitating with CspH43Q was only 31 Ϯ 11% of that co-immunoprecipitating with CspWT (p Ͻ 0.01, supplemental Fig.…”

Section: Csp Affects the Level Of Cftr Expression-our Laboratory Hasmentioning

confidence: 78%

“…reported earlier that overexpression of Csp produced a dosedependent reduction in mature (band C) CFTR and an increase in immature (band B) CFTR, which accumulated in the endoplasmic reticulum (ER) (22,23). To determine whether Csp was acting on CFTR present in vesicular carriers on their way from the endoplasmic reticulum to the Golgi apparatus or on CFTR still in the ER, we employed overexpression of Sar1-GTP (H79G), a constitutively active mutant of Sar1, as an independent means of blocking CFTR exit from the ER.…”

Section: Csp Affects the Level Of Cftr Expression-our Laboratory Hasmentioning

confidence: 98%

“…Our laboratory previously established a novel role for Csp at endoplasmic reticulum (ER) membranes in modulating the trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR). The overexpression of Csp blocked the maturation of CFTR (22,23), producing a dose-dependent reduction in mature (band C) CFTR and an increase in immature (band B) CFTR, which was localized to the ER. The Csp-induced block of CFTR maturation required Hsc70/Hsp70, because expression of a J-domain mutant of Csp (CspH43Q) that cannot stimulate Hsc70/Hsp70 ATPase activity allowed CFTR maturation.…”

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confidence: 98%

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Cysteine String Protein Promotes Proteasomal Degradation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by Increasing Its Interaction with the C Terminus of Hsp70-interacting Protein and Promoting CFTR Ubiquitylation

Schmidt

Watts

Aridor

et al. 2009

Journal of Biological Chemistry

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Cysteine string protein (Csp) is a J-domain-containing protein whose overexpression blocks the exit of cystic fibrosis transmembrane conductance regulator (CFTR) from the endoplasmic reticulum (ER). Another method of blocking ER exit, the overexpression of Sar1-GTP, however, yielded twice as much immature CFTR compared with Csp overexpression. This finding suggested that Csp not only inhibits CFTR ER exit but also facilitates the degradation of immature CFTR. This was confirmed by treatment with a proteasome inhibitor, which returned the level of immature CFTR to that found in cells expressing Sar1-GTP only. CspH43Q, which does not interact with Hsc70/Hsp70 efficiently, did not promote CFTR degradation, suggesting that the pro-degradative effect of Csp requires Hsc70/Hsp70 binding/activation. In agreement with this, Csp overexpression increased the amount of Hsc70/Hsp70 co-immunoprecipitated with CFTR, whereas overexpression of CspH43Q did not. The Hsc70/Hsp70 binding partner C terminus of Hsp70-interacting protein (CHIP) can target CFTR for proteasome-mediated degradation. Csp overexpression also increased the amount of CHIP co-immunoprecipitated with CFTR. In addition, CHIP interacted directly with Csp, which was confirmed by in vitro binding experiments. Csp overexpression also increased CFTR ubiquitylation and reduced the half-life of immature CFTR. These findings indicate that Csp not only regulates the exit of CFTR from the ER, but that this action is accompanied by Hsc70/ Hsp70 and CHIP-mediated CFTR degradation.Cysteine string protein (Csp, DnaJC5) 2 is a member of the DnaJ/Hsp40 protein chaperone family (1, 2). Csp contains a short N-terminal sequence, followed by the J-domain, a linker region, and the central cysteine-rich region, which gives these proteins their name. The cysteine residues in this region are post-translationally modified by the palmitate groups that serve for the membrane attachment of Csp (3). This structure is followed by a C-terminal domain, which is the most variable region among the three Csp paralogs in the human genome.Csp was originally discovered as an abundant protein in presynaptic junctions of Drosophila neurons (4). Csps are expressed at high levels in neurons and in cell types involved in regulated exocytosis (5-7), where they have been shown to govern exocytic secretory functions. For example, depolarizationinduced synaptic vesicle exocytosis is impaired in neurons from Csp-deficient Drosophila (8), and Csp overexpression produced decreases in stimulated insulin release from ␤-cells and stimulated catecholamine release from chromaffin cells (9 -11). Deletion of the Csp gene proved to be lethal both in Drosophila and in mice causing a progressive, fatal sensorimotor disorder characterized by developing neurodegenerative changes (12, 13).As for other Hsp40 homologs, the J-domain is responsible for the ability of Csp to bind Hsc70/Hsp70 and stimulate its ATPase activity (14). Similar to other chaperone proteins, Csp can be found in many different protein complexes in the...

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Section: Discussionsupporting

confidence: 80%