Exposing the subunit diversity within protein complexes: A mass spectrometry approach

Rozen, Shelly; Tieri, Alessandra; Ridner, Gabriela; Stark, Ann-Kathrin; Schmaler, Tilo; Ben‐Nissan, Gili; Dubiel, Wolfgang; Sharon, Michal

doi:10.1016/j.ymeth.2012.12.013

Cited by 31 publications

(38 citation statements)

References 48 publications

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“…CSN8 appeared in three different forms: two variants, which were separated in the pI dimension, displayed a migration pattern in the size dimension that was slower than that of the third species, reflecting their higher molecular weight. This result is in accordance with our previous observation, which indicated that CSN8, purified from human erythrocytes, is present in two forms, with a mass difference of 0.5 kDa, possibly resulting from two adjacent translation initiation sites (Met1 and Met6) (64).…”

Section: Resultssupporting

confidence: 93%

Dynamic Regulation of the COP9 Signalosome in Response to DNA Damage

Füzesi-Levi

Ben‐Nissan

Bianchi

et al. 2014

Molecular and Cellular Biology

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The COP9 signalosome (CSN) is an evolutionarily conserved protein complex that participates in the regulation of the ubiquitin/26S proteasome pathway by controlling the function of cullin-RING-ubiquitin ligases. Impressive progress has been made in deciphering its critical role in diverse cellular and developmental processes. However, little is known about the underlying regulatory principles that coordinate its function. Through biochemical and fluorescence microscopy analyses, we determined that the complex is localized in the cytoplasm, nucleoplasm, and chromatin-bound fractions, each differing in the composition of posttranslationally modified subunits, depending on its location within the cell. During the cell cycle, the segregation between subcellular localizations remains steady. However, upon UV damage, a dose-dependent temporal shuttling of the CSN complex into the nucleus was seen, accompanied by upregulation of specific phosphorylations within CSN1, CSN3, and CSN8. Taken together, our results suggest that the specific spatiotemporal composition of the CSN is highly controlled, enabling the complex to rapidly adapt and respond to DNA damage.

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Section: Resultssupporting

confidence: 93%

Dynamic Regulation of the COP9 Signalosome in Response to DNA Damage

Füzesi-Levi

Ben‐Nissan

Bianchi

et al. 2014

Molecular and Cellular Biology

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“…To evaluate the purity of the sample, SPOP (50 pmol) was loaded onto a monolithic column (Rozen et al , 2013) and eluted in a linear gradient of 20–50% ACN over 30 min. The protein eluted as a single peak at 17.6–19.8 min, at approximately 33% ACN .…”

Section: Resultsmentioning

confidence: 99%

Higher‐order oligomerization promotes localization of SPOP to liquid nuclear speckles

et al. 2016

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Membrane‐less organelles in cells are large, dynamic protein/protein or protein/RNA assemblies that have been reported in some cases to have liquid droplet properties. However, the molecular interactions underlying the recruitment of components are not well understood. Herein, we study how the ability to form higher‐order assemblies influences the recruitment of the speckle‐type POZ protein (SPOP) to nuclear speckles. SPOP, a cullin‐3‐RING ubiquitin ligase (CRL3) substrate adaptor, self‐associates into higher‐order oligomers; that is, the number of monomers in an oligomer is broadly distributed and can be large. While wild‐type SPOP localizes to liquid nuclear speckles, self‐association‐deficient SPOP mutants have a diffuse distribution in the nucleus. SPOP oligomerizes through its BTB and BACK domains. We show that BTB‐mediated SPOP dimers form linear oligomers via BACK domain dimerization, and we determine the concentration‐dependent populations of the resulting oligomeric species. Higher‐order oligomerization of SPOP stimulates CRL3SPOP ubiquitination efficiency for its physiological substrate Gli3, suggesting that nuclear speckles are hotspots of ubiquitination. Dynamic, higher‐order protein self‐association may be a general mechanism to concentrate functional components in membrane‐less cellular bodies.

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“…This was accomplished by combining MS-based proteomic identification of proteins extracted from gel bands, structural MS/MS analysis and a monolithic column-based approach. In brief, the monolithic column was used under denaturing conditions to separate the individual subunits of the isolated complexes 45 . The eluted flow from this column was split, using a Nanomate robot (see Methods).…”

Section: Resultsmentioning

confidence: 99%

“…The eluted flow from this column was split, using a Nanomate robot (see Methods). One fraction was sprayed directly into the mass spectrometer for accurate mass determination, while the second was collected for sequence identification following tryptic digestion, MS analysis and a database search 45 . This approach enabled us to relate the specific subunit sequences with the accurate masses (see Table 1 for subunit masses and identities).…”

Section: Resultsmentioning

confidence: 99%

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Regulation of focal adhesion formation by a vinculin-Arp2/3 hybrid complex

et al. 2014

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Focal adhesions (FAs) are large multi-protein complexes that act as transmembrane links between the extracellular matrix and the actin cytoskeleton. Recently, FAs were extensively characterized, yet the molecular mechanisms underlying their mechanical and signalling functions remain unresolved. To address this question, we isolated protein complexes containing different FA components, from chicken smooth muscle, and characterized their properties. Here we identified 'hybrid complexes' consisting of the actin-nucleating subunits of Arp2/3 and either vinculin or vinculin and a-actinin. We further show that suppression of p41-ARC, a central component of native Arp2/3, which is absent from the hybrid complexes, increases the levels of the Arp2/3-nucleating core in FA sites and stimulates FA growth and dynamics. In contrast, overexpression of p41-ARC adversely affects FAs. These results support the view that Arp2/3 can form modular 'hybrid complexes' containing an actinnucleating 'functional core', and 'anchoring domains' (vinculin/p41-ARC) that regulate its subcellular localization.

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Exposing the subunit diversity within protein complexes: A mass spectrometry approach

Cited by 31 publications

References 48 publications

Dynamic Regulation of the COP9 Signalosome in Response to DNA Damage

Dynamic Regulation of the COP9 Signalosome in Response to DNA Damage

Higher‐order oligomerization promotes localization of SPOP to liquid nuclear speckles

Regulation of focal adhesion formation by a vinculin-Arp2/3 hybrid complex

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