RESUMO
LMAN1 (ERGIC-53) is a key mammalian cargo receptor responsible for the export of a subset of glycoproteins from the endoplasmic reticulum. Together with its soluble coreceptor MCFD2, LMAN1 transports coagulation factors V (FV) and VIII (FVIII). Mutations in LMAN1 or MCFD2 cause the genetic bleeding disorder combined deficiency of FV and FVIII (F5F8D). The LMAN1 carbohydrate recognition domain (CRD) binds to both glycoprotein cargo and MCFD2 in a Ca(2+)-dependent manner. To understand the biochemical basis and regulation of LMAN1 binding to glycoprotein cargo, we solved crystal structures of the LMAN1-CRD bound to Man-α-1,2-Man, the terminal carbohydrate moiety of high mannose glycans. Our structural data, combined with mutagenesis and in vitro binding assays, define the central mannose-binding site on LMAN1 and pinpoint histidine 178 and glycines 251/252 as critical residues for FV/FVIII binding. We also show that mannobiose binding is relatively independent of pH in the range relevant for endoplasmic reticulum-to-Golgi traffic, but is sensitive to lowered Ca(2+) concentrations. The distinct LMAN1/MCFD2 interaction is maintained at these lowered Ca(2+) concentrations. Our results suggest that compartmental changes in Ca(2+) concentration regulate glycoprotein cargo binding and release from the LMAN1·MCFD2 complex in the early secretory pathway.
Assuntos
Retículo Endoplasmático/metabolismo , Fator VIII/metabolismo , Fator V/metabolismo , Lectinas de Ligação a Manose/metabolismo , Manose/metabolismo , Proteínas de Membrana/metabolismo , Animais , Sítios de Ligação/genética , Western Blotting , Células COS , Cálcio/metabolismo , Sequência de Carboidratos , Chlorocebus aethiops , Cristalografia por Raios X , Fator V/genética , Fator VIII/genética , Glicina/genética , Glicina/metabolismo , Complexo de Golgi/metabolismo , Histidina/genética , Histidina/metabolismo , Humanos , Manose/química , Lectinas de Ligação a Manose/química , Lectinas de Ligação a Manose/genética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMO
Mitochondria are essential organelles that harbor a reduced genome, and expression of that genome requires regulated metabolism of its transcriptome by nuclear-encoded proteins. Despite extensive investigation, a comprehensive map of the yeast mitochondrial transcriptome has not been developed and all of the RNA-metabolizing proteins have not been identified, both of which are prerequisites to elucidating the basic RNA biology of mitochondria. Here, we present a mitochondrial transcriptome map of the yeast S288C reference strain. Using RNAseq and bioinformatics, we show the expression level of all transcripts, revise all promoter, origin of replication, and tRNA annotations, and demonstrate for the first time the existence of alternative splicing, mirror RNAs, and a novel RNA processing site in yeast mitochondria. The transcriptome map has revealed new aspects of mitochondrial RNA biology and we expect it will serve as a valuable resource. As a complement to the map, we present our compilation of all known yeast nuclear-encoded ribonucleases (RNases), and a screen of this dataset for those that are imported into mitochondria. We sought to identify RNases that are refractory to recovery in traditional mitochondrial screens due to an essential function or eclipsed accumulation in another cellular compartment. Using this in silico approach, the essential RNase of the nuclear and cytoplasmic exosome, Dis3p, emerges as a strong candidate. Bioinformatics and in vivo analyses show that Dis3p has a conserved and functional mitochondrial-targeting signal (MTS). A clean and marker-less chromosomal deletion of the Dis3p MTS results in a defect in the decay of intron and mirror RNAs, thus revealing a role for Dis3p in mitochondrial RNA decay.