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A priority code in mitochondrial presequences - Data set 1: IPs from rabbit reticulocyte lysates

ABSTRACT: The biogenesis of mitochondria relies on the import of hundreds of different precursor proteins from the cytosol. Most of these proteins are synthesized with N-terminal presequences which serve as matrix targeting signals. Presequences consistently form amphipathic helices, but they considerably differ in respect to their primary sequence and length. Here we show that presequences can be grouped into different classes on basis of their specific features. Using a test set of different presequences, we observed that Class 1 presequences endow precursor proteins with improved in vitro import characteristics. We developed IQ-compete, a novel fluorescence de-quenching-based assay to monitor the import efficiencies of mitochondrial precursors in vivo. With this assay, we confirmed the increased import competence of Class 1 presequences. Using mass spectrometry, we found that the presequence of the Class 1 protein Oxa1 specifically recruits the tetratricopeptide repeat (TPR) containing protein TOMM34 to the cytosolic precursor protein. TOMM34 and its yeast homolog Cns1 apparently serve as presequence-specific targeting factors that increase the import efficiency of a specific subset of mitochondrial precursor proteins. Our results suggest that presequences contain a protein-specific priority code that encrypts the targeting mechanism of individual mitochondrial precursor proteins.

INSTRUMENT(S):

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

TISSUE(S): Cell Culture

SUBMITTER: Markus Räschle

LAB HEAD: Markus Räschle

PROVIDER: PXD053210 | Pride | 2025-07-09

REPOSITORIES: Pride

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			Action	DRS
	20240221_HF1_MaRa_SA_P0221_Atp5_01.raw	Raw
	20240221_HF1_MaRa_SA_P0221_Atp5_02.raw	Raw
	20240221_HF1_MaRa_SA_P0221_Atp5_03.raw	Raw
	20240221_HF1_MaRa_SA_P0221_DHFR_01.raw	Raw
	20240221_HF1_MaRa_SA_P0221_DHFR_02.raw	Raw

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Project description:Aim: To identify regulatory factors that control: (1) chloroplast protein importand (2) chloroplast-to-nucleus signalling. This project is a joint proposal from the Jarvis lab which is interested in chloroplast protein import [1] and the Moller lab which is interested in plastid-to-nucleus signalling [2]. Background: The majority of chloroplast proteins are encoded in the nucleus and imported post-translationally into chloroplasts. The abundance of chloroplast proteins may therefore be regulated at multiple levels. It is well documented that the nuclear gene expression is responsive to (largely unknown) signals from the chloroplast [23] and evidence is now emerging that protein import is also a regulated process [1]. Protein import into chloroplasts is mediated by protein complexes in the outer and inner envelope membranes called Toc and Tic respectively. Biochemical studies of pea chloroplasts identified several Toc/Tic components. These proteins are mechanistic or structural components of the import apparatus. Arabidopsis homologues of the pea Toc/Tic proteins were identified by the AGI. Pea Toc34 is represented in Arabidopsis by two genes "Toc33 and Toc34" and pea Toc75 is represented by three genes. These different Tocs have different expression patterns and are proposed to have different precursor protein recognition specificities. The factors that regulate Toc expression in concert with the needs of plastids in developmentally different cells are unknown. Proposal: Two Arabidopsis mutants will be analysed. The ppi1 mutant is null for the putative precursor protein receptor Toc33 [1]and the ppi3 mutant is null for a putative component of the protein import channel Toc75-IV (on chromosome IV). ppi1 plants are yellow-green in appearance but remarkably healthy and grow only slightly more slowly than wild type. By contrast ppi3 plants are indistinguishable from wild type by eye although analysis of the mutant's chloroplast proteome is beginning to reveal some differences (K. Lilley personal communication). Gene expression changes in ppi1 are likely to be quite extensive. Retardation of chloroplast development in ppi1 will activate retrograde signalling pathways so that many nuclear photosynthetic genes are down-regulated. Changes in the expression of photosynthetic genes and of the genes responsible for mediating these responses may therefore be observed. Any regulatory and signalling genes identified will be of interest to the Moller lab. The expression of factors that regulate Toc/Tic gene expression may also be altered in ppi1. It should be possible to distinguish these factors from those involved in the general control of chloroplast gene expression by comparing the results from the two mutants. Genes affected in both mutants are more likely to be involved in regulating chloroplast import since it is unlikely that widespread changes in gene expression will be observed in ppi3. Changes in the expression of factors that regulate import post-translationallyand of the Toc/Tic genes themselves (many are on the RNA) may also be observed. References: 1. Jarvis P. et al. (1998) Science 282: 100-103. 2. Moller S.G. et al. (2001) Genes Dev. 15:90-103. 3. Jarvis P. (2001) Curr. Biol. 11: R307-R310.

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A priority code in mitochondrial presequences - Data set 1: IPs from rabbit reticulocyte lysates

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