The Structure of the Human Centrin 2-Xeroderma Pigmentosum Group C Protein Complex

Thompson, James R.; Ryan, Zachary C.; Salisbury, Jeffrey L.; Kumar, Rajiv

doi:10.1074/jbc.m513667200

Cited by 87 publications

(129 citation statements)

References 55 publications

(54 reference statements)

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“…on April 7, 2019 by guest http://mcb.asm.org/ Similar structures have been identified in Kar1 and XPC/Rad4 (7,22,37,46,50). The interactions between Cdc31 and diverse cellular proteins were revealed in genetic studies (23) and are consistent with multiple roles.…”

supporting

confidence: 55%

Centrin/Cdc31 Is a Novel Regulator of Protein Degradation

Chen

Madura

2008

Molecular and Cellular Biology

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Rad23 is required for efficient protein degradation and performs an important role in nucleotide excision repair. Saccharomyces cerevisiae Rad23, and its human counterpart (hHR23), are present in a complex containing the DNA repair factor Rad4 (termed XPC, for xeroderma pigmentosum group C, in humans). XPC/ hHR23 was also reported to bind centrin-2, a member of the superfamily of calcium-binding EF-hand proteins. We report here that yeast centrin, which is encoded by CDC31, is similarly present in a complex with Rad4/Rad23 (called NEF2). The interaction between Cdc31 and Rad23/Rad4 varied by growth phase and reflected oscillations in Cdc31 levels. Strikingly, a cdc31 mutant that formed a weaker interaction with Rad4 showed sensitivity to UV light. Based on the dual function of Rad23, in both DNA repair and protein degradation, we questioned if Cdc31 also participated in protein degradation. We report here that Cdc31 binds the proteasome and multiubiquitinated proteins through its carboxy-terminal EF-hand motifs. Moreover, cdc31 mutants were highly sensitive to drugs that cause protein damage, failed to efficiently degrade proteolytic substrates, and formed altered interactions with the proteasome. These findings reveal for the first time a new role for centrin/Cdc31 in protein degradation.The microtubule organizing center regulates cytoskeletal integrity, and is termed the centrosome in higher eukaryotes, and the spindle pole-body (SPB) in yeast. The duplication of the SPB/centrosome is a key event that initiates entry into the cell cycle. The SPB is a proteinaceous structure that is embedded in the nuclear envelope, and its duplication marks exit from the G 1 phase and the onset of chromosome duplication. The composition of the SPB has been defined, and the steps that lead to its duplication have been studied extensively using biochemical, genetic, and microscopic methods (3,5,6,18,24,28,30,43,44). An important component of the SPB is Cdc31 (3, 13), which in yeast is encoded by the essential CDC31 gene. CDC31 was isolated as a suppressor of kar1, a mutant that is unable to duplicate the SPB at the nonpermissive temperature (6). As with kar1, defects in CDC31 (cdc31) also cause G 1 phase arrest, due to a failure in SPB duplication (48). Intriguingly, the kar1 defect was suppressed by Dsk2 (5), which has functional and structural resemblance to Rad23 (15,29,34,40).The three centrin proteins that are expressed in human cells (30, 31) display differential tissue-specific levels (27,36,49). However, all three isoforms share the key structural features that are present in EF-hand proteins (32). Human CEN3 is closely related to Cdc31 and can interfere with SPB duplication when overexpressed in yeast (30). An N-terminal regulatory domain is proposed to regulate the activity of the C-terminal domain, possibly through Ca 2ϩ -mediated signaling (42). The C terminus binds various cellular proteins (1, 7, 11, 12, 16, 19-23, 28, 37, 46, 50), although the effect of these interactions in regulating growth control is not well ...

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supporting

confidence: 55%

Centrin/Cdc31 Is a Novel Regulator of Protein Degradation

Chen

Madura

2008

Molecular and Cellular Biology

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“…S1B, Left). This result is consistent with previous data showing that XPC and RAD23B interact in a 1:1 ratio (26) and that XPC and CETN2 also interact in a 1:1 ratio (27,28). Furthermore, a 1:1:1 stoichiometry is consistent with the size of the ∼200-kDa product we observe for the cross-linked complex (Fig.…”

Section: Resultssupporting

confidence: 82%

Architecture of the human XPC DNA repair and stem cell coactivator complex

Zhang

Grob

et al. 2015

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

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The Xeroderma pigmentosum complementation group C (XPC) complex is a versatile factor involved in both nucleotide excision repair and transcriptional coactivation as a critical component of the NANOG, OCT4, and SOX2 pluripotency gene regulatory network. Here we present the structure of the human holo-XPC complex determined by single-particle electron microscopy to reveal a flexible, ear-shaped structure that undergoes localized loss of order upon DNA binding. We also determined the structure of the complete yeast homolog Rad4 holo-complex to find a similar overall architecture to the human complex, consistent with their shared DNA repair functions. Localized differences between these structures reflect an intriguing phylogenetic divergence in transcriptional capabilities that we present here. Having positioned the constituent subunits by tagging and deletion, we propose a model of key interaction interfaces that reveals the structural basis for this difference in functional conservation. Together, our findings establish a framework for understanding the structure-function relationships of the XPC complex in the interplay between transcription and DNA repair.transcription | stem cells | DNA repair | structure | biochemistry

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“…However, we also observed binding (albeit to a much lesser extent) of nonSUMOylated centrin-2 to XPC protein, in agreement with previous in vitro binding studies (Araki et al, 2001) and our notion that nuclear localization of centrin-2 was not completely abolished upon interference with the SUMOylation pathway. Structural studies using small peptides showed residues 847-863 of the XPC protein to associate with centrin-2 in vitro (Popescu et al, 2003;Thompson et al, 2006). However, these experiments did not address the posttranslational modifications on either XPC protein or centrin-2.…”

Section: Discussionmentioning

confidence: 93%

SUMO-dependent regulation of centrin-2

Klein

Nigg

2009

Journal of Cell Science

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2+-binding proteins that are highly conserved from yeast to humans. Centrin-2 is a core component of the centrosome of higher eukaryotes. In addition, it is present within the nucleus, in which it is part of the xeroderma pigmentosum group C (XPC) complex, which controls nucleotide excision repair (NER). Regulation of the subcellular distribution of centrin-2 has so far remained elusive. Here we show that centrin-2 is a substrate of SUMOylation in vitro and in vivo, and that it is preferentially modified by SUMO2/3. Moreover, we identify the SUMO E3-like ligase human polycomb protein 2 (PC2; also known as hPC2) as essential for centrin-2 modification. Interference with the SUMOylation pathway leads to a striking defect in nuclear localization of centrin-2 and accumulation in the cytoplasm, whereas centrosomal recruitment of centrin-2 is unaffected. Depletion of the XPC protein mimics this situation and we provide evidence that SUMO conjugation of centrin-2 enhances its binding to the XPC protein. These data show that the nucleocytoplasmic shuttling of centrin-2 depends on the SUMO system and indicates that localization of centrin-2 within the nucleus depends on its ability to bind to the XPC protein.Supplementary material available online at

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The Structure of the Human Centrin 2-Xeroderma Pigmentosum Group C Protein Complex

Cited by 87 publications

References 55 publications

Centrin/Cdc31 Is a Novel Regulator of Protein Degradation

Centrin/Cdc31 Is a Novel Regulator of Protein Degradation

Architecture of the human XPC DNA repair and stem cell coactivator complex

SUMO-dependent regulation of centrin-2

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