RESUMEN
Multisubunit Tethering Complexes (MTCs) are a set of conserved protein complexes that tether vesicles at the acceptor membrane. Interactions with other components of the trafficking machinery regulate MTCs through mechanisms that are partially understood. Here, we systematically investigate the interactome that regulates MTCs. We report that P4-ATPases, a family of lipid flippases, interact with MTCs that participate in the anterograde and retrograde transport at the Golgi, such as TRAPPIII. We use the P4-ATPase Drs2 as a paradigm to investigate the mechanism and biological relevance of this interplay during transport of Atg9 vesicles. Binding of Trs85, the sole-specific subunit of TRAPPIII, to the N-terminal tail of Drs2 stabilizes TRAPPIII on membranes loaded with Atg9 and is required for Atg9 delivery during selective autophagy, a role that is independent of P4-ATPase canonical functions. This mechanism requires a conserved I(S/R)TTK motif that also mediates the interaction of the P4-ATPases Dnf1 and Dnf2 with MTCs, suggesting a broader role of P4-ATPases in MTC regulation.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Relacionadas con la Autofagia/genética , Proteínas Relacionadas con la Autofagia/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , ATPasas Transportadoras de Calcio/química , ATPasas Transportadoras de Calcio/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismoRESUMEN
Human mitochondrial transcription factor A, TFAM, is essential for mitochondrial DNA packaging and maintenance and also has a crucial role in transcription. Crystallographic analysis of TFAM in complex with an oligonucleotide containing the mitochondrial light strand promoter (LSP) revealed two high-mobility group (HMG) protein domains that, through different DNA recognition properties, intercalate residues at two inverted DNA motifs. This induced an overall DNA bend of ~180°, stabilized by the interdomain linker. This U-turn allows the TFAM C-terminal tail, which recruits the transcription machinery, to approach the initiation site, despite contacting a distant DNA sequence. We also ascertained that structured protein regions contacting DNA in the crystal were highly flexible in solution in the absence of DNA. Our data suggest that TFAM bends LSP to create an optimal DNA arrangement for transcriptional initiation while facilitating DNA compaction elsewhere in the genome.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Proteínas Mitocondriales/química , Proteínas Mitocondriales/metabolismo , Conformación de Ácido Nucleico , Regiones Promotoras Genéticas , Estructura Terciaria de Proteína , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Cristalografía por Rayos X , ADN/química , ADN/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Motivos de NucleótidosRESUMEN
The regulation of mitochondrial DNA (mtDNA) processes is slowly being characterized at a structural level. We present here crystal structures of human mitochondrial regulator mTERF, a transcription termination factor also implicated in replication pausing, in complex with double-stranded DNA oligonucleotides containing the tRNA(Leu)(UUR) gene sequence. mTERF comprises nine left-handed helical tandem repeats that form a left-handed superhelix, the Zurdo domain.