The protein chaperone Ssa1 affects mRNA localization to the mitochondria

Abstract

Edited by Ulrike KutayKeywords: mRNA localization Mitochondria Hsp70 chaperone Ssa1 Tom70 Yeast a b s t r a c t Many nuclear-transcribed mRNAs encoding mitochondrial proteins are localized near the mitochondrial outer membrane. A yet unresolved question is whether protein synthesis is important for transport of these mRNAs to their destination. Herein we present a connection between mRNA localization in yeast and the protein chaperone Ssa1. Ssa1 depletion lowered mRNA association with mitochondria while its overexpression increased it. A genome-wide analysis revealed that Ssa proteins preferentially affect mRNAs encoding hydrophobic proteins, which are expected targets for these protein chaperones. Importantly, deletion of the mitochondrial receptor Tom70 abolished the impact of Ssa1 overexpression on mRNAs encoding Tom70 targets. Taken together, our results suggest a role for Ssa1 in mediating localization of nascent peptide-ribosome-mRNA complexes to the mitochondria, consistent with a co-translational transport process.

Localized translation near the mitochondrial outer membrane: An update

“…An analysis of cells with reduced levels of this protein revealed a decreased association between mitochondria and certain mRNAs, many of which encode proteins with a high hydrophobic score. 50 These results were consistent with a role for this Hsp70 protein in the folding of hydrophobic domains. Indeed, Ssa1 was shown to interact with RNCs, 77 suggesting that its impact on the localization of mRNAs occurs during their translation.…”

“…Recent studies have revealed the molecular players that are involved in both of these modes (see Section 3). 30,31,50,51 Ribosomes' association with mitochondria can be measured by western analysis of mitochondrial fractions with an antibody that recognizes a ribosomal protein. 31,52 This technique, however, should be interpreted cautiously, as mitochondrial fractions usually contain ER fragments, which also contain ribosomes.…”

“…Indeed, Ssa1 was shown to interact with RNCs, 77 suggesting that its impact on the localization of mRNAs occurs during their translation. Importantly, Ssa1 is a cytosolic chaperone that is absent from the mitochondrial fraction, 50 therefore its effects take place during mRNA transport to the mitochondria.…”

Isolation of mRNAs Associated with Yeast Mitochondria to Study Mechanisms of Localized Translation

“…Furthermore, translated features such as the AUG, mitochondrial targeting sequence (MTS) or ORF regions were shown to assist in localization 8,11,15 . Hsp70-family protein chaperone and protein receptor on the mitochondria outer membrane were also shown to support mRNA association, further implying that encoded-protein features are important for mRNA localization 16 . This is consistent with a model in which the ribosome-emerging protein serves as recognition element for targeting mRNA-ribosome-protein complex to the mitochondria 17 .…”

“…Centrifuge spheroplasts at 3,000 x g for 5 min at 4 °C and discard the supernatant. 16. Wash twice with cold Mannitol Buffer.…”