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Ric-8 proteins are molecular chaperones that direct nascent G protein ? subunit membrane association.

ABSTRACT: Ric-8A (resistance to inhibitors of cholinesterase 8A) and Ric-8B are guanine nucleotide exchange factors that enhance different heterotrimeric guanine nucleotide-binding protein (G protein) signaling pathways by unknown mechanisms. Because transgenic disruption of Ric-8A or Ric-8B in mice caused early embryonic lethality, we derived viable Ric-8A- or Ric-8B-deleted embryonic stem (ES) cell lines from blastocysts of these mice. We observed pleiotropic G protein signaling defects in Ric-8A(-/-) ES cells, which resulted from reduced steady-state amounts of G?(i), G?(q), and G?(13) proteins to <5% of those of wild-type cells. The amounts of G?(s) and total G? protein were partially reduced in Ric-8A(-/-) cells compared to those in wild-type cells, and only the amount of G?(s) was reduced substantially in Ric-8B(-/-) cells. The abundances of mRNAs encoding the G protein ? subunits were largely unchanged by loss of Ric-8A or Ric-8B. The plasma membrane residence of G proteins persisted in the absence of Ric-8 but was markedly reduced compared to that in wild-type cells. Endogenous G?(i) and G?(q) were efficiently translated in Ric-8A(-/-) cells but integrated into endomembranes poorly; however, the reduced amounts of G protein ? subunits that reached the membrane still bound to nascent G??. Finally, G?(i), G?(q), and G?(1) proteins exhibited accelerated rates of degradation in Ric-8A(-/-) cells compared to those in wild-type cells. Together, these data suggest that Ric-8 proteins are molecular chaperones required for the initial association of nascent G? subunits with cellular membranes.

SUBMITTER: Gabay M

PROVIDER: S-EPMC3870195 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature

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Ric-8 proteins are molecular chaperones that direct nascent G protein α subunit membrane association.

Gabay Meital M Pinter Mary E ME Wright Forrest A FA Chan PuiYee P Murphy Andrew J AJ Valenzuela David M DM Yancopoulos George D GD Tall Gregory G GG

Science signaling 20111122 200

Ric-8A (resistance to inhibitors of cholinesterase 8A) and Ric-8B are guanine nucleotide exchange factors that enhance different heterotrimeric guanine nucleotide-binding protein (G protein) signaling pathways by unknown mechanisms. Because transgenic disruption of Ric-8A or Ric-8B in mice caused early embryonic lethality, we derived viable Ric-8A- or Ric-8B-deleted embryonic stem (ES) cell lines from blastocysts of these mice. We observed pleiotropic G protein signaling defects in Ric-8A(-/-) E ...[more]

PMID: 22114146

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Project description:Aim and experimental set-up: Sequencing of small RNA from total RNA fractions. The aim of this experiment was to compare profiles of miRNA expression between the following genetic backgrounds: Col-0 (wild type), era1-2 (farnesyl transferase mutant), j2-2/j3-2 + pJ3:J3 (j2/j3 double knockout expressing transgenic J3 under the control of the endogenous J3 promoter), j2-2/j3-2 + pJ3:J3C417S (j2/j3 double knockout expressing transgenic J3 mutated in the farnesylation site (C417S) under the control of the endogenous J3 promoter). Seedlings were grown under sterile conditions for 16 days. Two biological replicates of each genotype were harvested, and total RNA was prepared by Trizol extraction. Small RNA libraries were constructed using NEBNext Small RNA Library Prep Set and sequenced on an Illumina platform. Sequencing of small RNA bound to AGO1 in membrane fractions The aim of this experiment was to characterize AGO1-bound small RNAs in membrane fractions, and to answer two specific questions: Can miRNAs be identified whose association with membrane-bound AGO1 is different between the following four genotypes: Col-0 (wild type), era1-2 (farnesyl transferase mutant), j2-2/j3-2 + pJ3:J3 (j2/j3 double knockout expressing transgenic J3 under the control of the endogenous J3 promoter), j2-2/j3-2 + pJ3:J3C417S (j2/j3 double knockout expressing transgenic J3 mutated in the farnesylation site (C417S) under the control of the endogenous J3 promoter) Is the ratio between reads matching miRNA and miRNA* different between the above four genotypes? Seedlings were grown under sterile conditions for 16 days. Two biological replicates of each genotype were harvested. Microsomes were prepared from 2g of seedling tissue, solubilized by 1% deoxycholate and AGO1 was immunoprecipitated with specific antibodies (Agrisera). Immunopurified AGO1 was eluted from Protein A sepharose beads by competitive elution with antigenic AGO1 peptide. Small RNA was extracted by Trizol extraction from eluted AGO1. Small RNA libraries were constructed using NEBNext Small RNA Library Prep Set and sequenced on an Illumina platform.

Dataset Information

Ric-8 proteins are molecular chaperones that direct nascent G protein ? subunit membrane association.

Publications

Ric-8 proteins are molecular chaperones that direct nascent G protein α subunit membrane association.

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