RESUMEN
A multifunctional calcium-binding protein, centrin-1, is specifically expressed in male germ cells, certain neurons and ciliated cells. We identified centrin-1 as a protein interacting with SUMO-2/3 using yeast two-hybrid screening of a mouse testicular cDNA library. In bead halo assays, the interaction between centrin-1 and SUMO-2/3 was reduced in the presence of EGTA and facilitated by the addition of CaCl2. immunostaining of seminiferous tubules in 35-day-old mouse testes revealed that cells in the layer containing spermatogonia showed colocalization of SUMO-2/3 with centrin-1 in cytoplasmic spots. Identification of centrin-1 as the EGTA-sensitive SUMO-2/3-interacting protein indicates the possible role of calcium in modulating the centrin-1-SUMO-2/3 interaction and suggests the importance of this interaction in mouse testis.
Asunto(s)
Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/metabolismo , Calcio/metabolismo , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/metabolismo , Motivos EF Hand , Túbulos Seminíferos/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Espermatogonias/metabolismo , Animales , Proteínas de Unión al Calcio/genética , Centrosoma/metabolismo , Quelantes/química , Proteínas Cromosómicas no Histona/genética , Ácido Egtácico/química , Biblioteca de Genes , Células HeLa , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Transporte de Proteínas , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/metabolismo , Túbulos Seminíferos/citología , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/genética , Espermatogonias/citología , Ubiquitinas/genética , Ubiquitinas/metabolismoRESUMEN
SUMO-interacting motifs (SIMs) play a central role in the fate of SUMO-modified proteins. Here we report a real-time SUMO-binding assay. It can be applied to the identification of SIMs and to screening for the identification of novel SUMO-binding proteins. Using this assay, we investigated the SIMs in SETDB1 and MCAF1 to gain insight into the assembly of SETDB1-MCAF1-mediated gene silencing.
Asunto(s)
Mapeo de Interacción de Proteínas/métodos , Proteína SUMO-1/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , N-Metiltransferasa de Histona-Lisina , Humanos , Unión Proteica , Proteína Metiltransferasas/química , Proteína Metiltransferasas/metabolismo , Proteínas Represoras , Factores de Tiempo , Factores de Transcripción/química , Factores de Transcripción/metabolismoRESUMEN
The small ubiquitin-related modifier 2/3 (SUMO2/3) can be post-translationally conjugated to a wide variety of proteins constituting chromatin, the platform for genetic and epigenetic regulation. Nevertheless, it is unclear how SUMO2/3 and SUMO2/3-modified proteins are delivered to the chromatin fibers. Here we report that the largest subunit of chromatin assembly factor 1 (CAF-1), human p150, interacts directly and preferentially with SUMO2/3. Amino acid residue of 98-105 in p150 is essential and sufficient for SUMO2/3 interaction. p150-SUMO2/3 interaction coincided with regions that replicate chromatin fibers, because accumulation of the proliferating cell nuclear antigen (PCNA), and incorporation of bromodeoxyuridine (BrdU) were detected at foci co-localized with both p150 and SUMO2/3 during the S-phase in a cell line expressing epitope-tagged p150. Although inhibition of SUMO2/3 expression had only a small effect on p150 deposition on the replication sites, depletion of p150 led to delocalization of SUMO2/3 from the replication foci. Furthermore, p150 mutants deficient in SUMO2/3 interaction, caused a major reduction of SUMO2/3 at the replication foci. Thus, our findings suggest an expanded role of p150 as a SUMO2/3-interacting factor, and raise the intriguing possibility that p150 plays a role in promoting delivery of SUMO2/3 or SUMO2/3-modified proteins (or both) on chromatin fibers during replication.