Folding of Fumarase during Mitochondrial Import Determines its Dual Targeting in Yeast

Sass, Ehud; Karniely, Sharon; Pines, Ophry

doi:10.1074/jbc.m302344200

Cited by 79 publications

(85 citation statements)

References 28 publications

(45 reference statements)

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“…Although fumarase cannot be imported post-translationally, in this study, we show that in vivo fumarase is exposed to TEV protease in the cytosol during import. This result is consistent with the requirement for cytosolic chaperones of the Ssa (Hsp70) family for fumarase import in vivo, suggesting that they interact with the protein in the cytosol prior to or during translocation (14,18). This is also consistent with translation-coupled import in vitro, in which the presequence of fumarase is accessible to cleavage by externally added mitochondrial processing peptidase en route to mitochondria (14).…”

Section: Discussionsupporting

confidence: 84%

“…It is important to point out that when expressed in yeast, these two fumarase derivatives are fully imported into mitochondria and do not distribute between the cytosol and mitochondria (not shown). This is expected, because, as we have previously reported, changes within the fumarase mature sequence or the addition of certain tags abolishes the fumarase dual subcellular distribution (18). This has allowed us to examine fumarase in the context of translocation without the complications of dual targeting of the protein.…”

Section: Tev Protease Can Be Targeted To the Cytosol Or Tomentioning

confidence: 61%

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Translation-coupled Translocation of Yeast Fumarase into Mitochondria in Vivo

Yogev

Karniely

Pines

2007

Journal of Biological Chemistry

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Fumarase represents proteins that cannot be imported into mitochondria after the termination of translation (post-translationally). Utilizing mitochondrial and cytosolic versions of the tobacco etch virus (TEV) protease, we show that mitochondrially targeted fumarase harboring a TEV protease recognition sequence is efficiently cleaved by the mitochondrial but not by the cytosolic TEV protease. Nonetheless, fumarase was readily cleaved by cytosolic TEV when its import into mitochondria was slowed down by either (i) disrupting the activity of the TOM complex, (ii) lowering the growth temperature, or (iii) reducing the inner membrane electrochemical potential. Accessibility of the fumarase nascent chain to TEV protease under such conditions was prevented by low cycloheximide concentrations, which impede translation. In addition, depletion of the ribosome-associated nascent polypeptide-associated complex (NAC) reduced the fumarase rate of translocation into mitochondria and exposed it to TEV cleavage in the cytosol. These results indicate that cytosolic exposure of the fumarase nascent chain depends on both translocation and translation rates, allowing us to discuss the possibility that import of fumarase into mitochondria occurs while the ribosome is still attached to the nascent chain.

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Section: Discussionsupporting

confidence: 84%

Section: Tev Protease Can Be Targeted To the Cytosol Or Tomentioning

confidence: 61%

Translation-coupled Translocation of Yeast Fumarase into Mitochondria in Vivo

Yogev

Karniely

Pines

2007

Journal of Biological Chemistry

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“…It is known that a single transla- tional product of the FUM1 gene, encoding fumarase, is distributed between the cytosol and the mitochondria (37). These two isoenzymes of fumarase are encoded by the same gene, and mature cytosolic and mitochondrial fumarase isoenzymes are identical (38). Nevertheless, we detected two isoforms in the 2D gels, probably indicating that fumarase is post-translationally modified to a further extent.…”

Section: Proteinmentioning

confidence: 69%

Comparative Proteome Analysis of Saccharomyces cerevisiae Grown in Chemostat Cultures Limited for Glucose or Ethanol

Kolkman

Olsthoorn

Heeremans

et al. 2005

Molecular & Cellular Proteomics

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The use of chemostat culturing enables investigation of steady-state physiological characteristics and adaptations to nutrient-limited growth, while all other relevant growth conditions are kept constant. We examined and compared the proteomic response of wild-type Saccharomyces cerevisiae CEN.PK113-7D to growth in aerobic chemostat cultures limited for carbon sources being either glucose or ethanol. To obtain a global overview of changes in the proteome, we performed triplicate analyses using two-dimensional gel electrophoresis and identified proteins of interest using MS. Relative quantities of about 400 proteins were obtained and analyzed statistically to determine which protein steady-state expression levels changed significantly under glucose-or ethanollimited conditions. Interestingly, only enzymes involved in central carbon metabolism showed a significant change in steady-state expression, whereas expression was only detected in one of both carbon source-limiting conditions for 15 of these enzymes. Side effects that were previously reported for batch cultivation conditions, such as responses to continuous variation of specific growth rate, to carbon-catabolite repression, and to accumulation of toxic substrates, were not observed. Moreover, by comparing our proteome data with corresponding mRNA data, we were able to unravel which processes in the central carbon metabolism were regulated at the level of the proteome, and which processes at the level of transcriptome. Importantly, we show here that the combined approach of chemostat cultivation and comprehensive proteome analysis allowed us to study the primary effect of single limiting conditions on the yeast proteome. Molecular & Cellular Proteomics 4:1-11, 2005. Two-dimensional (2D)1 gel electrophoresis is a powerful tool to visualize hundreds of proteins at a time, which in combination with MS leads to their identification. This technology has been applied to the yeast Saccharomyces cerevisiae for the large-scale identification of more than 400 proteins, resulting in yeast reference maps (1-7). Other S. cerevisiae studies used 2D gel electrophoresis to obtain an overview of global changes in the yeast proteome as function of stimuli such as cadmium (8), lithium (9), H 2 O 2 (10), or sorbic acid (11).Most of these differential proteome studies on S. cerevisiae were performed on cells cultured in batch mode, i.e. in shake flasks or in reactors. Batch cultivation makes use of a closed system, in which all nutrients are in excess at the start of the cultivation. In terms of microbial physiology, such batch cultivation is relatively poorly controlled, because the composition of the growth medium and consequently the growth rate changes continuously. The yeast cells take up nutrients from the media while metabolites are excreted in the culture system, and their growth arrests when one of the nutrients is depleted or when too many toxic substrates are accumulated. The high concentration of carbon source in the culture, which is essential for this type of cultu...

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“…In yeast, the fumarase (FUM1) gene product, encoding a mitochondrial presequence, is distributed to both the mitochondrion and the cytosol. Once imported and processed within the mitochondrion, the protein relies on particular folding to facilitate its re-export to the cytosol through the translocation pore [45].…”

Section: Acknowledgmentsmentioning

confidence: 99%

Plant organellar protein targeting: a traffic plan still under construction

Mackenzie

2005

Trends in Cell Biology

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It has long been understood that specific features of a protein and its corresponding import apparatus dictate the behavior of mitochondrial proteins in their intracellular targeting behavior. In plants, the process by which proteins are directed to organelles has been influenced uniquely by the introduction to the cell of plastids. Parallel functions carried out within the mitochondrion and plastid permit the sharing of proteins and emergence of mechanisms to facilitate dual-targeting of the nuclear-encoded products to both compartments. These include transcriptional and translational variations, relaxation of translation initiation controls and conditional cellular influences. Details of the dual targeting system are emerging from recent studies, and evidence of variation in protein targeting behavior across plant families and across organisms implies that the system itself is in flux. This trend towards multi-targeting enhances protein versatility across eukaryotes -one means of cellular response to developmental or environmental influence.

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Folding of Fumarase during Mitochondrial Import Determines its Dual Targeting in Yeast

Cited by 79 publications

References 28 publications

Translation-coupled Translocation of Yeast Fumarase into Mitochondria in Vivo

Translation-coupled Translocation of Yeast Fumarase into Mitochondria in Vivo

Comparative Proteome Analysis of Saccharomyces cerevisiae Grown in Chemostat Cultures Limited for Glucose or Ethanol

Plant organellar protein targeting: a traffic plan still under construction

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