RESUMEN
BACKGROUND: RNA is often targeted to be localized to the specific subcellular compartments. Specific localization of mRNA is believed to be an important mechanism for targeting their protein products to the locations, where their function is required. RESULTS: In this study we performed the genome wide transcriptome analysis of peroxisome preparations from the mouse liver using microarrays. We demonstrate that RNA is absent inside peroxisomes, however it is associated at their exterior via the noncovalent contacts with the membrane proteins. We detect enrichment of specific sets of transcripts in two preparations of peroxisomes, purified with different degrees of stringency. Importantly, among these were mRNAs encoding bona fide peroxisomal proteins, such as peroxins and peroxisomal matrix enzymes involved in beta-oxidation of fatty acids and bile acid biosynthesis. The top-most enriched mRNA, whose association with peroxisomes we confirm microscopically was Hmgcs1, encoding 3-hydroxy-3-methylglutaryl-CoA synthase, a crucial enzyme of cholesterol biosynthesis pathway. We observed significant representation of mRNAs encoding mitochondrial and secreted proteins in the peroxisomal fractions. CONCLUSIONS: This is a pioneer genome-wide study of localization of mRNAs to peroxisomes that provides foundation for more detailed dissection of mechanisms of RNA targeting to subcellular compartments.
Asunto(s)
Estudio de Asociación del Genoma Completo , Peroxisomas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Animales , Perfilación de la Expresión Génica , Espacio Intracelular , Espectrometría de Masas , Ratones , Transporte de ARN , TranscriptomaRESUMEN
The ionotropic 5HT(3) receptor was expressed in transiently transfected mammalian cells, yielding an unprecedented high concentration of up to 12 million receptors per cell. Receptor traffic in the plasma membrane of live cells was observed continuously over 24 h by fluorescence scanning confocal microscopy. This was possible by using 5HT(3) receptor-specific fluorescent ligands with high binding affinity and low off-rate to pulse label receptors at any time after appearance on the cell surface, and label subsequently those receptors expressed later by another, spectrally distinguishable, high-affinity fluorescent ligand. Having reached a critical cell surface concentration of approximately 3000 receptors/microm(2), the receptors started to aggregate in patches with a 4-fold increased surface concentration. The clusters were constantly delivered from a pool of freshly expressed receptors isotropically distributed within the basolateral region of the cell membrane. From there, they migrated to and accumulated on the apical cell surface approximately 9 h after transfection. Individual clusters grew until they reached a critical size of 1-2 microm when they merged to form with 3-5 microm large macroclusters. Clustered receptors were immobile on the minute time scale but always coexisted with monomeric receptors in the regions surrounding the clusters as revealed by fluorescence correlation spectroscopy. Because the receptor density of 12 000 receptors/microm(2) in the patches is as high as that found in two-dimensional crystals of certain membrane proteins, such patches might be a proper source for direct crystallization of membrane proteins without prior purification.
Asunto(s)
Canales Iónicos/metabolismo , Agregación de Receptores , Receptores de Serotonina/biosíntesis , Receptores de Serotonina/metabolismo , Línea Celular , Membrana Celular/genética , Membrana Celular/metabolismo , Fluoresceína/metabolismo , Colorantes Fluorescentes/metabolismo , Regulación de la Expresión Génica , Humanos , Canales Iónicos/genética , Microscopía Confocal , Transporte de Proteínas/genética , Ensayo de Unión Radioligante , Agregación de Receptores/genética , Receptores de Serotonina/genética , Receptores de Serotonina 5-HT3 , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rodaminas/metabolismo , Espectrometría de Fluorescencia , TransfecciónRESUMEN
We used differential screening of cDNAs from individual taste receptor cells to identify candidate taste transduction elements in mice. Among the differentially expressed clones, one encoded Trpm5, a member of the mammalian family of transient receptor potential (TRP) channels. We found Trpm5 to be expressed in a restricted manner, with particularly high levels in taste tissue. In taste cells, Trpm5 was coexpressed with taste-signaling molecules such as alpha-gustducin, Ggamma13, phospholipase C-beta2 (PLC-beta2) and inositol 1,4,5-trisphosphate receptor type III (IP3R3). Our heterologous expression studies of Trpm5 indicate that it functions as a cationic channel that is gated when internal calcium stores are depleted. Trpm5 may be responsible for capacitative calcium entry in taste receptor cells that respond to bitter and/or sweet compounds.