Dynamics and retention of misfolded proteins in native ER membranes

Nehls, Sarah; Snapp, Erik L.; Cole, Nelson B.; Zaal, Kristien J.M.; Kenworthy, Anne K.; Roberts, Theresa H; Ellenberg, Jan; Presley, John F.; Siggia, Eric D.; Lippincott‐Schwartz, Jennifer

doi:10.1038/35010558

Cited by 250 publications

(269 citation statements)

References 37 publications

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“…3A), a small region of the cell Crt-GFP fluorescence was repeatedly photobleached. Over time, Crt-GFP fluorescence in the entire cell was steadily and uniformly lost, whereas fluorescence of adjacent cells was unaffected, as expected for a freely diffusing protein distributed throughout a continuous ER (14,16,18).…”

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confidence: 56%

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Monitoring chaperone engagement of substrates in the endoplasmic reticulum of live cells

Snapp

Sharma

Lippincott‐Schwartz

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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The folding environment in the endoplasmic reticulum (ER) depends on multiple abundant chaperones that function together to accommodate a range of substrates. The ways in which substrate engagement shapes either specific chaperone dynamics or general ER attributes in vivo remain unknown. In this study, we have evaluated how changes in substrate flux through the ER influence the diffusion of both the lectin chaperone calreticulin and an inert reporter of ER crowdedness. During acute changes in substrate load, the inert probe revealed no changes in ER organization, despite significant changes in calreticulin dynamics. By contrast, inhibition of the lectin chaperone system caused rapid changes in the ER environment that could be reversed over time by easing new substrate burden. Our findings provide insight into the normal organization and dynamics of an ER chaperone and characterize the capacity of the ER to maintain homeostasis during acute changes in chaperone activity and availability.castanospermine ͉ fluorescence loss in photobleaching ͉ fluorescence recovery after photobleaching ͉ pactamycin ͉ puromycin

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confidence: 56%

“…Thus, the diffusion of ER-GFP fluorescence can be used to monitor changes in the capacity of an ER component to sample its environment (16,18).…”

mentioning

confidence: 99%

Monitoring chaperone engagement of substrates in the endoplasmic reticulum of live cells

Snapp

Sharma

Lippincott‐Schwartz

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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110

112

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“…We first performed a FRAP (Nehls et al, 2000) experiment on the PDI-GFP-expressing egg chambers (Figure 7, A-D). Because PDI-GFP is resident in the ER, it can be used as a marker for diffusion in this organelle.…”

Section: A Very Active Transport Out Of the Ermentioning

confidence: 99%

mRNA Localization and ER-based Protein Sorting Mechanisms Dictate the Use of Transitional Endoplasmic Reticulum-Golgi Units Involved in Gurken Transport inDrosophilaOocytes

Herpers

Rabouille

2004

MBoC

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The anteroposterior and dorsoventral axes of the future embryo are specified within Drosophila oocytes by localizing gurken mRNA, which targets the secreted Gurken transforming growth factor-␣ synthesis and transport to the same site. A key question is whether gurken mRNA is targeted to a specialized exocytic pathway to achieve the polar deposition of the protein. Here, we show, by (immuno)electron microscopy that the exocytic pathway in stage 9 -10 Drosophila oocytes comprises a thousand evenly distributed transitional endoplasmic reticulum (tER)-Golgi units. Using Drosophila mutants, we show that it is the localization of gurken mRNA coupled to efficient sorting of Gurken out of the ER that determines which of the numerous equivalent tER-Golgi units are used for the protein transport and processing. The choice of tER-Golgi units by mRNA localization makes them independent of each other and represents a nonconventional way, by which the oocyte implements polarized deposition of transmembrane/secreted proteins. We propose that this pretranslational mechanism could be a general way for targeted secretion in polarized cells, such as neurons. INTRODUCTIONPolarized localization of proteins is achieved by at least two different mechanisms. The first one relies on restricted localization of mRNAs that encode cytosolic proteins, allowing local protein translation, thus creating differential protein activity and generating cell asymmetry and polarity (Bashirullah et al., 1998). This is, for instance, the case of actin mRNA localization in the nerve terminal of neurons (Lee and Hollenbeck, 2003), oskar localization at the posterior pole of Drosophila oocytes (Ephrussi et al., 1991), and ash1 localization in dividing yeast cells (Long et al., 1997). On the other hand, the mechanism ensuring the polarized deposition of transmembrane or secreted proteins is thought, in mammalian cells, to be achieved by posttranslational sorting events in the trans-Golgi network (TGN). There, signals in their cytoplasmic tail or lumenal domain are deciphered, resulting in the directed movement of specialized transport vesicles to allow specific deposition at the apical or basolateral plasma membrane of epithelial cells (Ikonen and Simons, 1998;Nelson and Yeaman, 2001).However, there is a growing number of transmembrane and secreted proteins for which their transcript also exhibits a restricted localization. The transcript for the yeast plasma membrane protein Ist2 is actively transported along the acto-myosin network to the bud tip (Takizawa et al., 2000), and the protein is deposited locally (Juschke et al., 2004). wingless mRNA is localized apically in epithelial cells (Simmonds et al., 2001). In a stage 9 -10 Drosophila oocytes, gurken mRNA is transported and deposited exclusively in the dorsal/anterior corner (D/A; Neuman-Silberberg and Schupbach, 1993; Figure 1B). gurken encodes a protein that is synthesized in the endoplasmic reticulum (ER) as a 285-amino acid type I transmembrane protein precursor and transported to the Golgi apparat...

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“…Filter trap analysis of cell lysates after 1% SDS treatment indicated that the G345E mutant forms SDS-resistant large structures in the presence of MG-132 ( Figure 4E). Because FRAP analysis has been used to analyze the mobility of misfolded proteins (Nehls et al, 2000;Kim et al, 2002), we used this technique to assess the behavior of the Y37C mutant (increased insolubility; Figures 1, C and E, and 4, A and B) at the centrosome. Even in the absence of the proteasome inhibitor, the fluorescence recovery of this mutant was ϳ70%, whereas the fluorescence recoveries of the R518 mutant (unaffected for degradation and insolubility) and the wild-type protein were both 90% (Figure 4, C and D).…”

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confidence: 99%

MKKS Is a Centrosome-shuttling Protein Degraded by Disease-causing Mutations via CHIP-mediated Ubiquitination

Hirayama

Yamazaki²,

Kitamura³

et al. 2008

MBoC

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McKusick-Kaufman syndrome (MKKS) is a recessively inherited human genetic disease characterized by several developmental anomalies. Mutations in the MKKS gene also cause Bardet-Biedl syndrome (BBS), a genetically heterogeneous disorder with pleiotropic symptoms. However, little is known about how MKKS mutations lead to disease. Here, we show that disease-causing mutants of MKKS are rapidly degraded via the ubiquitin-proteasome pathway in a manner dependent on HSC70 interacting protein (CHIP), a chaperone-dependent ubiquitin ligase. Although wild-type MKKS quickly shuttles between the centrosome and cytosol in living cells, the rapidly degraded mutants often fail to localize to the centrosome. Inhibition of proteasome functions causes MKKS mutants to form insoluble structures at the centrosome. CHIP and partner chaperones, including heat-shock protein (HSP)70/heat-shock cognate 70 and HSP90, strongly recognize MKKS mutants. Modest knockdown of CHIP by RNA interference moderately inhibited the degradation of MKKS mutants. These results indicate that the MKKS mutants have an abnormal conformation and that chaperone-dependent degradation mediated by CHIP is a key feature of MKKS/BBS diseases. INTRODUCTIONMcKusick-Kaufman syndrome (MKKS) is a recessively inherited human genetic disease predominantly characterized by developmental anomalies, including vaginal atresia with hydrometrocolpos, polydactyly, and congenital heart defects (McKusick et al., 1964). The MKKS gene encodes a 570-amino acid polypeptide with weak but significant similarity to group II chaperonins . Mutations in the MKKS gene also cause Bardet-Biedl syndrome (BBS), a genetically heterogeneous disorder characterized by obesity, retinal dystrophy, polydactyly, mental retardation, renal malformation, and hypogenitalism (Beales et al., 1999), and thus MKKS is also called BBS6. The MKKS mRNA is widely expressed in various tissues, including those affected by MKKS and BBS diseases . Although more than 10 mutations in the MKKS gene have been identified in patients (Beales et al., 2001;Slavotinek et al., 2002), little is known about how they cause MKKS and BBS.Twelve of the genes that are responsible for BBS (BBS1-12) have been identified so far, and the products of several of these genes have been suggested to be involved in intraflagellar transport in cilia and intracellular trafficking in the cytosol Badano et al., 2005;Beales, 2005;Blacque and Leroux, 2006). Cargo-carrying motors play crucial roles in intraflagellar and intracellular transport systems by bidirectionally moving on microtubules, and BBS4 interacts with the dynactin complex, which mediates interactions between cargoes and the dynein motor (Kim et al., 2004). Retrograde transport of melanosomes in zebrafish is slowed by knockdown of BBS2 or BBS4-8 (Yen et al., 2006), whereas mutations in Caenorhabditis elegans BBS7 and BBS8 cause delays in intraflagellar transport driven by kinesin motors (Ou et al., 2005). The Chlamydomonas homologue of BBS5 is essential for flagellum formation (Li et al., ...

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Dynamics and retention of misfolded proteins in native ER membranes

Cited by 250 publications

References 37 publications

Monitoring chaperone engagement of substrates in the endoplasmic reticulum of live cells

Monitoring chaperone engagement of substrates in the endoplasmic reticulum of live cells

mRNA Localization and ER-based Protein Sorting Mechanisms Dictate the Use of Transitional Endoplasmic Reticulum-Golgi Units Involved in Gurken Transport inDrosophilaOocytes

MKKS Is a Centrosome-shuttling Protein Degraded by Disease-causing Mutations via CHIP-mediated Ubiquitination

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