ER-associated retrograde SNAREs and the Dsl1 complex mediate an alternative, Sey1p-independent homotypic ER fusion pathway

Rogers, Jason V.; McMahon, Conor; Baryshnikova, Anastasia; Hughson, Frederick M.; Rose, Mark D.

doi:10.1091/mbc.e14-07-1220

Cited by 16 publications

(16 citation statements)

References 48 publications

(88 reference statements)

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“…When wild-type δ-COP was replaced by the HARS double mutant to abolish the δ-COP:Dsl1p interaction, yeast grew normally. This result is not unexpected, given the previous finding that the Dsl1p lasso is functionally redundant with another region of Dsl1p, the highly conserved C-terminal E domain (9, 24), for COPI-mediated transport: Yeast containing a deletion of either the entire lasso region or of the E-domain grow normally (24). Strikingly, yeast lacking the Dsl1p E domain-and therefore rendered dependent on the Dsl1 lasso for COPI-dependent transport and hence viability-displayed marked growth-rate defects when lasso binding was compromised by the δ-COP HARS mutations (Fig.…”

Section: Resultssupporting

confidence: 52%

Structural basis for the binding of tryptophan-based motifs by δ-COP

Suckling

Poon

Travis

et al. 2015

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

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Coatomer consists of two subcomplexes: the membrane-targeting, ADP ribosylation factor 1 (Arf1):GTP-binding βγδζ-COP F-subcomplex, which is related to the adaptor protein (AP) clathrin adaptors, and the cargo-binding αβ'e-COP B-subcomplex. We present the structure of the C-terminal μ-homology domain of the yeast δ-COP subunit in complex with the WxW motif from its binding partner, the endoplasmic reticulum-localized Dsl1 tether. The motif binds at a site distinct from that used by the homologous AP μ subunits to bind YxxΦ cargo motifs with its two tryptophan residues sitting in compatible pockets. We also show that the Saccharomyces cerevisiae Arf GTPase-activating protein (GAP) homolog Gcs1p uses a related WxxF motif at its extreme C terminus to bind to δ-COP at the same site in the same way. Mutations designed on the basis of the structure in conjunction with isothermal titration calorimetry confirm the mode of binding and show that mammalian δ-COP binds related tryptophan-based motifs such as that from ArfGAP1 in a similar manner. We conclude that δ-COP subunits bind Wx n(1-6) [WF] motifs within unstructured regions of proteins that influence the lifecycle of COPI-coated vesicles; this conclusion is supported by the observation that, in the context of a sensitizing domain deletion in Dsl1p, mutating the tryptophan-based motif-binding site in yeast causes defects in both growth and carboxypeptidase Y trafficking/processing. C OPI vesicles mediate retrograde trafficking from the Golgi to the endoplasmic reticulum (ER) and within the Golgi (reviewed in refs. 1-3). The COPI vesicle coat consists of two major components, coatomer and the small GTPase ADP ribosylation factor 1 (Arf1). Coatomer is an ∼600 kDa heteroheptameric complex consisting of two linked subcomplexes, the βγδζ-COP F-subcomplex and the αβ'e-COP B-subcomplex, all conserved from yeast to humans.Recent studies have led to a model for COPI-coated vesicle formation in which the F-subcomplex functions as a traditional Arf1:GTP effector but with two membrane-attached Arf1:GTP molecules binding to quasi-equivalent sites on a single F-subcomplex, recruiting F-subcomplex and its associated B-subcomplex onto the membrane en bloc (4). Once the vesicle has budded from its donor membrane, an Arf GTPase-activating protein (ArfGAP) catalyzes the hydrolysis of GTP on Arf1, and the Arf1:GDP dissociates from the membrane, followed later by the dissociation of the coatomer complex that was bound to the transport vesicle (5). ArfGAPs also have been proposed to function at different stages in vesicle biogenesis, both as terminators and, during an earlier cargoediting step, as effectors (reviewed in depth in ref. 6). In the final stages of its life, a COPI-coated vesicle docks with the ER via the Dsl1-tethering complex, completes uncoating, and finally undergoes SNARE-mediated fusion with the ER (7-9).The two main ArfGAPs associated with COPI-dependent retrograde transport in yeast are Gcs1p and Glo3p. These proteins can substitute for one another, at least partially,...

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Section: Resultssupporting

confidence: 52%

Structural basis for the binding of tryptophan-based motifs by δ-COP

Suckling

Poon

Travis

et al. 2015

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

Self Cite

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“…To test whether the signalsequence-containing reporter proteins would also localize to the ER membrane in cells lacking lipid droplets, their subcellular localization was analyzed in an inducible strain in which three of the genes required for neutral lipid synthesis (ARE1, ARE2 and DGA1) were deleted and the fourth gene was placed under transcriptional control of a glucose repressible promoter (GAL-LRO1). Under conditions where LRO1 is repressed, Erg6 and Tgl1(Δtm) and the signal-sequence-containing reporters ss-Erg6 and ss-Tgl1(Δtm), displayed a circular staining that merged with the localization of the ER luminal marker protein ss-mCherry-HDEL, indicating that signal-sequence-containing proteins relocalize to the ER in cells that have no lipid droplets (Rogers et al, 2014) (Fig. 1B).…”

Section: Resultsmentioning

confidence: 99%

Mature lipid droplets are accessible to ER luminal proteins

Mishra

Khaddaj

Cottier

et al. 2016

Journal of Cell Science

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Lipid droplets are found in most organisms where they serve to store energy in the form of neutral lipids. They are formed at the endoplasmic reticulum (ER) membrane where the neutral-lipidsynthesizing enzymes are located. Recent results indicate that lipid droplets remain functionally connected to the ER membrane in yeast and mammalian cells to allow the exchange of both lipids and integral membrane proteins between the two compartments. The precise nature of the interface between the ER membrane and lipid droplets, however, is still ill-defined. Here, we probe the topology of lipid droplet biogenesis by artificially targeting proteins that have high affinity for lipid droplets to inside the luminal compartment of the ER. Unexpectedly, these proteins still localize to lipid droplets in both yeast and mammalian cells, indicating that lipid droplets are accessible from within the ER lumen. These data are consistent with a model in which lipid droplets form a specialized domain in the ER membrane that is accessible from both the cytosolic and the ER luminal side.

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“…These data illuminate a non‐canonical pathway to extracellular release mediated by a SNAP23‐dependent mechanism, suggesting a novel route for the release of tau from the neuron. SNARE proteins coordinate vesicle trafficking and exocytosis in all cells (Morelli et al , ; Rogers et al , ; Dubuke et al , ; Kuster et al , ; Zhu et al , ), and chaperones play an important role in several of these processes (Joglekar & Hay, ; Sharma et al , ). This has a number of important implications for understanding the propagation of pathological protein aggregates found in several neurodegenerative diseases.…”

Section: Discussionmentioning

confidence: 99%

DnaJ/Hsc70 chaperone complexes control the extracellular release of neurodegenerative‐associated proteins

et al. 2016

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It is now known that proteins associated with neurodegenerative disease can spread throughout the brain in a prionlike manner. However, the mechanisms regulating the trans-synaptic spread propagation, including the neuronal release of these proteins, remain unknown. The interaction of neurodegenerative diseaseassociated proteins with the molecular chaperone Hsc70 is well known, and we hypothesized that much like disaggregation, refolding, degradation, and even normal function, Hsc70 may dictate the extracellular fate of these proteins. Here, we show that several proteins, including TDP-43, a-synuclein, and the microtubule-associated protein tau, can be driven out of the cell by an Hsc70 cochaperone, DnaJC5. In fact, DnaJC5 overexpression induced tau release in cells, neurons, and brain tissue, but only when activity of the chaperone Hsc70 was intact and when tau was able to associate with this chaperone. Moreover, release of tau from neurons was reduced in mice lacking the DnaJC5 gene and when the complement of DnaJs in the cell was altered. These results demonstrate that the dynamics of DnaJ/Hsc70 complexes are critically involved in the release of neurodegenerative disease proteins.

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ER-associated retrograde SNAREs and the Dsl1 complex mediate an alternative, Sey1p-independent homotypic ER fusion pathway

Cited by 16 publications

References 48 publications

Structural basis for the binding of tryptophan-based motifs by δ-COP

Structural basis for the binding of tryptophan-based motifs by δ-COP

Mature lipid droplets are accessible to ER luminal proteins

DnaJ/Hsc70 chaperone complexes control the extracellular release of neurodegenerative‐associated proteins

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