RESUMO
The superfamily of metallothioneins (MTs) combines a diverse group of metalloproteins, sharing the characteristics of rather low molecular weight and high cysteine content. The latter provides MTs with the capability to coordinate thiophilic metal ions, in particular those with a d 10 electron configuration. The sub-family of plant MT3 proteins is only poorly characterized and there is a complete lack of three-dimensional structure information. Building upon our previous results on the Musa acuminata MT3 (musMT3) protein, the focus of the present work is to understand the metal cluster formation process, the role of the single histidine residue present in musMT3, and the metal ion binding affinity. We concentrate our efforts on the coordination of ZnII and CdII ions, using CoII as a spectroscopic probe for ZnII binding. The overall protein-fold is analysed with a combination of limited proteolytic digestion, mass spectrometry, and dynamic light scattering. Histidine coordination of metal ions is probed with extended X-ray absorption fine structure spectroscopy and CoII titration experiments. Initial experiments with isothermal titration calorimetry provide insights into the thermodynamics of metal ion binding.
Assuntos
Cádmio/metabolismo , Metalotioneína/metabolismo , Musa/química , Proteínas de Plantas/metabolismo , Zinco/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Cobalto/metabolismo , Complexos de Coordenação/química , Histidina/metabolismo , Hidrólise , Metalotioneína/química , Metalotioneína/genética , Mutação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Ligação ProteicaRESUMO
The Fanconi anemia (FA) core complex promotes the tolerance/repair of DNA damage at stalled replication forks by catalyzing the monoubiquitination of FANCD2 and FANCI. Intriguingly, the core complex component FANCM also catalyzes branch migration of model Holliday junctions and replication forks in vitro. Here we have characterized the ortholog of FANCM in fission yeast Fml1 in order to understand the physiological significance of this activity. We show that Fml1 has at least two roles in homologous recombination-it promotes Rad51-dependent gene conversion at stalled/blocked replication forks and limits crossing over during mitotic double-strand break repair. In vitro Fml1 catalyzes both replication fork reversal and D loop disruption, indicating possible mechanisms by which it can fulfill its pro- and antirecombinogenic roles.
Assuntos
Quebras de DNA de Cadeia Dupla , DNA Helicases/metabolismo , Reparo do DNA , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Troca Genética , DNA Helicases/genética , Replicação do DNA , DNA Cruciforme , DNA Fúngico/química , DNA Fúngico/genética , DNA Fúngico/metabolismo , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Conversão Gênica , Genes Fúngicos , Humanos , Mutação , Recombinação Genética , Proteínas de Schizosaccharomyces pombe/genéticaRESUMO
The sorting of post-Golgi R-SNAREs (vesicle-associated membrane protein (VAMP)1, 2, 3, 4, 7 and 8) is still poorly understood. To address this, we developed a system to investigate their localization, trafficking and cell-surface levels. Here, we show that the distribution and internalization of VAMPs 3 and 8 are determined solely through a new conserved mechanism that uses coiled-coil interactions, and that VAMP4 does not require these interactions for its trafficking. We propose that VAMPs 3 and 8 are trafficked while in a complex with Q-SNAREs. We also show that the dileucine motif of VAMP4 is required for both its internalization and retrieval to the trans-Golgi network. However, when the dileucine motif is mutated, the construct can still be internalized potentially through coiled-coil interactions with Q-SNAREs.