Recombinant Heptameric Coatomer Complexes: Novel Tools to Study Isoform‐Specific Functions

Sahlmüller, Monika; Strating, Jeroen R.P.M.; Beck, Rainer; Eckert, Priska; Popoff, Vincent; Haag, Mathias; Hellwig, Andrea; Berger, Imre; Brügger, Britta; Wieland, Felix

doi:10.1111/j.1600-0854.2011.01177.x

Cited by 25 publications

(28 citation statements)

References 41 publications

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“…Invitrogen Sf9 cells were tested by the manufacturer for contamination of bacteria, yeast, mycoplasma and virus and were characterized by isozyme and karyotype analysis. We used a baculoviral expression system essentially as described previously (Sahlmüller et al, 2011) with a ‘One-Strep-Tag’ at the C-terminus of α-COP. Recombinant S. cerevisiae myristoylated Arf1, and human nucleotide exchange factor ARNO, were purified from E. coli as described previously (Chardin et al, 1996; Randazzo et al, 1992).…”

Section: Methodsmentioning

confidence: 99%

9Å structure of the COPI coat reveals that the Arf1 GTPase occupies two contrasting molecular environments

Dodonova

Aderhold

Kopp

et al. 2017

eLife

104

150

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COPI coated vesicles mediate trafficking within the Golgi apparatus and between the Golgi and the endoplasmic reticulum. Assembly of a COPI coated vesicle is initiated by the small GTPase Arf1 that recruits the coatomer complex to the membrane, triggering polymerization and budding. The vesicle uncoats before fusion with a target membrane. Coat components are structurally conserved between COPI and clathrin/adaptor proteins. Using cryo-electron tomography and subtomogram averaging, we determined the structure of the COPI coat assembled on membranes in vitro at 9 Å resolution. We also obtained a 2.57 Å resolution crystal structure of βδ-COP. By combining these structures we built a molecular model of the coat. We additionally determined the coat structure in the presence of ArfGAP proteins that regulate coat dissociation. We found that Arf1 occupies contrasting molecular environments within the coat, leading us to hypothesize that some Arf1 molecules may regulate vesicle assembly while others regulate coat disassembly.DOI: http://dx.doi.org/10.7554/eLife.26691.001

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Section: Methodsmentioning

confidence: 99%

9Å structure of the COPI coat reveals that the Arf1 GTPase occupies two contrasting molecular environments

Dodonova

Aderhold

Kopp

et al. 2017

eLife

104

150

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“…Recombinant coatomer protein was expressed and purified, as described in Sahlmuller et al, (2011). In short, Sf9 insect cells were infected with baculovirus encoding for heptameric coatomer.…”

Section: Methodsmentioning

confidence: 99%

Arf1/COPI machinery acts directly on lipid droplets and enables their connection to the ER for protein targeting

Wilfling

Thiam

Olarte

et al. 2014

eLife

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249

274

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Lipid droplets (LDs) are ubiquitous organelles that store neutral lipids, such as triacylglycerol (TG), as reservoirs of metabolic energy and membrane precursors. The Arf1/COPI protein machinery, known for its role in vesicle trafficking, regulates LD morphology, targeting of specific proteins to LDs and lipolysis through unclear mechanisms. Recent evidence shows that Arf1/COPI can bud nano-LDs (∼60 nm diameter) from phospholipid-covered oil/water interfaces in vitro. We show that Arf1/COPI proteins localize to cellular LDs, are sufficient to bud nano-LDs from cellular LDs, and are required for targeting specific TG-synthesis enzymes to LD surfaces. Cells lacking Arf1/COPI function have increased amounts of phospholipids on LDs, resulting in decreased LD surface tension and impairment to form bridges to the ER. Our findings uncover a function for Arf1/COPI proteins at LDs and suggest a model in which Arf1/COPI machinery acts to control ER-LD connections for localization of key enzymes of TG storage and catabolism.DOI: http://dx.doi.org/10.7554/eLife.01607.001

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“…Recombinant coatomer (;1.3 mg/mL) (Sahlmuller et al 2011) or the respective buffer was co-injected with 1:10 diluted Alexa488-labeled secondary antibodies (Invitrogen) into stable HeLa cells using a FemtoJet microinjection device (Eppendorf) mounted on an Axiovert 35 microscope (Zeiss). Three to 6 h after microinjection, cells were transfected with a siRNA against COPB1 (Ambion, 24546) and induced for reporter protein expression 4 h posttransfection.…”

Section: Microinjection Of Recombinant Coatomermentioning

confidence: 99%

Phenotypic profiling of the human genome reveals gene products involved in plasma membrane targeting of SRC kinases

Ritzerfeld¹,

Remmele²,

Wang³

et al. 2011

Genome Res.

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SRC proteins are non-receptor tyrosine kinases that play key roles in regulating signal transduction by a diverse set of cell surface receptors. They contain N-terminal SH4 domains that are modified by fatty acylation and are functioning as membrane anchors. Acylated SH4 domains are both necessary and sufficient to mediate specific targeting of SRC kinases to the inner leaflet of plasma membranes. Intracellular transport of SRC kinases to the plasma membrane depends on microdomains into which SRC kinases partition upon palmitoylation. In the present study, we established a live-cell imaging screening system to identify gene products involved in plasma membrane targeting of SRC kinases. Based on siRNA arrays and a human model cell line expressing two kinds of SH4 reporter molecules, we conducted a genome-wide analysis of SH4-dependent protein targeting using an automated microscopy platform. We identified and validated 54 gene products whose down-regulation causes intracellular retention of SH4 reporter molecules. To detect and quantify this phenotype, we developed a software-based image analysis tool. Among the identified gene products, we found factors involved in lipid metabolism, intracellular transport, and cellular signaling processes. Furthermore, we identified proteins that are either associated with SRC kinases or are related to various known functions of SRC kinases such as other kinases and phosphatases potentially involved in SRC-mediated signal transduction. Finally, we identified gene products whose function is less defined or entirely unknown. Our findings provide a major resource for future studies unraveling the molecular mechanisms that underlie proper targeting of SRC kinases to the inner leaflet of plasma membranes.

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Recombinant Heptameric Coatomer Complexes: Novel Tools to Study Isoform‐Specific Functions

Cited by 25 publications

References 41 publications

9Å structure of the COPI coat reveals that the Arf1 GTPase occupies two contrasting molecular environments

9Å structure of the COPI coat reveals that the Arf1 GTPase occupies two contrasting molecular environments

Arf1/COPI machinery acts directly on lipid droplets and enables their connection to the ER for protein targeting

Phenotypic profiling of the human genome reveals gene products involved in plasma membrane targeting of SRC kinases

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