Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M310160200 on November 3, 2003

J. Biol. Chem., Vol. 279, Issue 6, 3956-3979, February 6, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/6/3956    most recent
M310160200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ohlmeier, S.
Right arrow Articles by Bergmann, U.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ohlmeier, S.
Right arrow Articles by Bergmann, U.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

The Yeast Mitochondrial Proteome, a Study of Fermentative and Respiratory Growth*

Steffen Ohlmeier{ddagger}, Alexander J. Kastaniotis, J. Kalervo Hiltunen, and Ulrich Bergmann

From the Biocenter Oulu and Department of Biochemistry, P. O. Box 3000, University of Oulu, Oulu FIN-90014, Finland

Received for publication, September 12, 2003 , and in revised form, November 3, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Saccharomyces cerevisiae is able to switch from fermentation to respiration (diauxic shift) with major changes in metabolic activity. This phenomenon has been previously studied on the transcriptional level. Here we present a parallel analysis of the yeast mitochondrial proteome and the corresponding transcriptional activity in cells grown on glucose (fermentation) and glycerol (respiration). A two-dimensional reference gel for this organelle proteome was established (available at www.biochem.oulu.fi/proteomics/), which contains about 800 intense spots. From 459 spots 253 individual proteins were identified, among them low abundant and hydrophobic proteins, and 37 proteins previously deemed hypothetical, with partially unknown cellular localization. After the diauxic shift, mitochondrial levels of only 18 proteins were changed (17 increased, with 1 decreased), among them proteins involved in the tricarboxylic acid cycle (Sdh1p, Sdh2p, and Sdh4p) and the respiratory chain (Cox4p, Cyb2p, and Qcr7p), proteins contributing to other respiratory pathways (Ach1p, Adh2p, Ald4p, Cat2p, Icl2p, and Pdh1p), and two proteins with unknown function (Om45p and Ybr230p). Apart from an overall increase in mitochondrial protein mass, the mitochondrial proteome remains remarkably constant, even in a major metabolic adaptation. This seemingly disagrees with results of the DNA microarray analyses, where a rather heterogenous up- or down-regulation of genes encoding mitochondrial proteins implies large changes in the proteome. We propose that the discrepancy between proteome and transcriptional regulation, apart from different translation efficiency, indicates a changed turnover rate of proteins in different physiological conditions.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Mitochondria have been known for a long time as the organelles responsible for the energy metabolism of eukaryotic cells. More recent studies suggest an additional role of the mitochondrion in apoptosis and aging (1–6). Likewise, numerous diseases, e.g. Alzheimer's disease, Parkinson's disease, Friedreich ataxia, diabetes mellitus, and malignant tumors (4, 7–14), were connected to mutations in genes coding for mitochondrial proteins. These findings have motivated increasing efforts to study the putative role of mitochondrial proteins in pathological events (11, 15).

The majority of mitochondrial proteins are nuclear encoded and targeted to the organelle by weakly conserved mitochondrial targeting sequences (MTS)1 (16–18). The MTS has been used as a marker for the prediction of mitochondrial proteomes from genomic databases. Currently, the expected proteome size of the yeast mitochondrion varies between 400 and 786 proteins (18–20). The mitochondrial proteome of higher eukaryotes is significantly larger, e.g. about 1000 proteins for the human mitochondrion (21). The reason for this increased proteome size of higher eukaryotes is not fully understood. A possible explanation offers the presence of isoforms for several mitochondrial enzymes and additional mitochondrial functions (22).

Elucidating the function and regulatory networks of proteins is one of the main challenges in the post-genomic era. Changes in the proteome under different environmental conditions, e.g. nutrient limitation or stress, can provide a starting point for the analysis of function of proteins. Thus far, studies of the mitochondrial proteome by two-dimensional gel electrophoresis have been described only for some higher eukaryotes (11, 22–29), but not for yeast. Because its genome is fully sequenced and its cell physiology has been exhaustively investigated, Saccharomyces cerevisiae is one of the most suitable eukaryotic model organism. Due to the fact that most genes coding for mitochondrial proteins are highly conserved among eukaryotes (22, 29, 30), it can serve as a model for mitochondrial physiology at the protein level.

S. cerevisiae can grow either with fermentative or respiratory metabolism. Glucose in abundance is catabolized mainly by fermentation (31). As glucose becomes exhausted, a transient growth arrest occurs. During this arrest, called the diauxic shift, the metabolism is changed from fermentation to respiration and adapted to alternative carbon sources, e.g. ethanol, glycerol, and oleic acid (32). This diauxic shift has been studied extensively at the mRNA level (32–37), and changes in transcription of genes involved in the tricarboxylic acid cycle and respiratory chain (37, 38), the gluconeogenesis and glyoxylate cycle (34, 39), as well as peroxisome biogenesis and {beta}-oxidation of fatty acids (35, 36, 39) have been revealed. However, only little is known how the diauxic shift affects the protein level (32, 34). The only proteomic studies on this subject reported so far have been focused on whole cell extracts (32, 34) and limited to highly abundant mitochondrial proteins (40). Thus far, it appears that transcriptional regulation is only partly transmitted to the protein level (41–44).

The aim of the present work was to achieve a more detailed characterization of the yeast mitochondrial proteome to study the diauxic shift at the proteomic level. This allows the investigation of how the transcriptional regulation, reported for this metabolic switch, is reflected at the protein level.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 REFERENCES
 
Preparation of Mitochondria—For the analysis of the fermentative growth the S. cerevisiae wild type strain BJ1991 (45) was cultivated on rich YPD (1% (w/v) yeast extract, 2% (w/v) peptone, and 2% (w/v) D-glucose) and collected at an optical density (600 nm) of 1.1. The glucose concentration of the culture media was monitored. For the study of the respiration a wild type culture was grown in YPD to an optical density (600 nm) of 1–2, pelleted, and transferred to YPG containing 3% (v/v) glycerol instead of D-glucose. After 16 h cells were collected, and mitochondria were prepared as described by Meisinger et al. (46).

For two-dimensional gel electrophoresis the mitochondrial pellet was resuspended in urea buffer (8 M urea, 2 M thiourea, 1% (w/v) CHAPS, 20 mM 1,4-dithio-DL-threitol, 0.8% (v/v) carrier ampholytes 3–10, 100 mM Tris-HCl, pH 7.5, 1 mM EDTA, 14 µM phenylmethylsulfonyl fluoride). The protein concentration was determined with a commercially available kit (RotiNanoquant, C. Roth, Karlsruhe, Germany) according to the method of Bradford (47), and protein aliquots (100 and 500 µg) were stored at –20 °C.

For transcriptional analyses the RNA was isolated from collected cells using the hot acidic phenol extraction method (84). The RNA was then further purified using the Qiagen RNeasy kit, following the RNA cleanup protocol provided by the manufacturer. The RNA was visually inspected on ethidium bromide-stained agarose gel to confirm that it was not degraded.

Two-dimensional Gel Electrophoresis and Protein Identification— The protein solution was adjusted with urea buffer to a final volume of 350 µl. Proteins were then transferred into IPG strips (pH 3–10, nonlinear, 18 cm, Amersham Biosciences, Uppsala, Sweden) overnight by in-gel rehydration. Isoelectric focusing was carried out at 20 °C with the Multiphor II system (Amersham Biosciences) under paraffin oil for 55 kVh (500 V 500 Vh, 500 V 2500 Vh, 3500 V 10 kVh, 3500 V 42 kVh) (48). Prior to separation in the second dimension the strips were equilibrated according to Görg et al. (49). The electrophoresis was done overnight in polyacrylamide gels (12.5%) with the Ettan DALT II system (Amersham Biosciences) at 1–2 W per gel and 12 °C. The gels were silver-stained (50) and analyzed with the two-dimensional PAGE image analysis software Melanie 3.0 (GeneBio, Geneva, Switzerland). The apparent isoelectric points (pI) and molecular masses of the proteins were calculated with Melanie 3.0 (GeneBio) using identified proteins with known parameters as a reference. The same software was used for the comparison of fermentative and respiratory protein pattern. An expression change was considered significant if the intensity of the corresponding spot differed reproducibly more than 3-fold. Changes in protein abundance were compared with the transcriptional data of the same samples. Excised spots were digested in-gel and identified from the peptide fingerprints as described elsewhere (85). The identification of a protein was accepted if the peptides (mass tolerance, 20 ppm) covered at least 30% of the complete sequence. A sequence coverage between 30 and 20% or a sequence coverage below 20% for protein fragments was only accepted if at least three main peaks of the mass spectrum matched with the sequence and the number of weak intensity peaks was clearly reduced. Monoclonal antibodies were obtained as a gift from Alexander Tzagoloff (anti-Kgd1p), Benedikt Westermann (anti-Aco1p), and Bernard D. Lemire (anti-Sdh2p).

Microarray Hybridization and Data Analysis—Experimental procedures for GeneChip were performed according to the Affymetrix Gene-Chip Expression Analysis Technical Manual. In essence, using 10 µg of total RNA as template double-stranded DNA was synthesized by means of the Superscript Choice System (Invitrogen, Rockville, MD) and T7-(dT)24 primer. The DNA was purified using GeneChip Sample Cleanup Module (Qiagen, Valencia, CA). In vitro transcription was performed to produce biotin-labeled cRNA using a BioArray HighYield RNA Transcription Labeling Kit (Enzo Diagnostics, Farmingdale, NY) according to the manufacturer's instructions. Biotinylated cRNA was cleaned with an GeneChip Sample Cleanup Module (Qiagen), fragmented to 35–200 nucleotides, and hybridized to Affymetrix Yeast genome S98 array, which contains ~6400 S. cerevisiae genes. After being washed, the array was stained with streptavidin-phycoerythrin (Molecular Probes, Eugene, OR). Staining signal was amplified by biotinylated anti-streptavidin (Vector Laboratories, Burlingame, CA) and second staining with streptavidin-phycoerythrin, and then scanned on HP GeneArray scanner. The expression data was analyzed using Affymetrix MicroArray Suite version 5.0. Signal intensities of all probe sets were scaled to the target value of 500.

Analysis of Protein Function and Sequence—Information about function, subcellular localization, and specific parameters of the identified proteins were obtained from the databases SWISS-PROT (www.expasy.org/sprot/), MIPS (mips.gsf.de/proj/yeast/CYGD/db/index.html), and SGD (genome-www.stanford.edu/Saccharomyces/). Duplication of genes was analyzed with the Yeast Gene Duplications data base (acer.gen.tcd.ie/~khwolfe/yeast/nova/index.html). The subcellular localization and the putative MTS cleavage site of mitochondrial proteins were predicted by TargetP (www.cbs.dtu.dk/services/TargetP/), PSORT II (psort.nibb.ac.jp/form2.html), and MITO-PROT (www.mips.biochem.mpg.de/cgi-bin/proj/medgen/mitofilter). The expected position (pI and molecular mass) of the protein spot within the two-dimensional gel was calculated with Compute pI/Mw (us.expasy.org/tools/pi_tool.html). Grand average of hydropathicity index (GRAVY) scores were predicted with SOSUI (sosui.proteome.bio.tuat.ac.jp/sosuiframe0.html).


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
REFERENCES
 
Mitochondria were purified from yeast cultures grown on a sufficient glucose concentration (at the beginning of the culture time 2% glucose and 1.2% glucose at the time of cell collection) to maintain glucose repression (Refs. 33 and 46, see below) and the protein fraction separated by two-dimensional gel electrophoresis. This resulted in about 800 intense spots detectable in a silver-stained two-dimensional gel. A two-dimensional pattern (reference gel) characteristic for fermentative growth, obtained from five mitochondrial preparations, is shown in Fig. 1. The corresponding two-dimensional gels specific for respiratory metabolism were obtained from seven mitochondrial preparations after growth on glycerol. Analyses of the detected proteins by tryptic digestion and mass spectrometry analyses (see "Experimental Procedures" for details) allowed the identification of 253 proteins from 459 spots (Table I).






View larger version (420K):
[in this window]
[in a new window]
  		FIG. 1.
Two-dimensional reference map of S. cerevisiae wild type BJ1991 mitochondria characteristic for fermentation. The mitochondrial proteins (100 µg) from a glucose grown yeast culture (optical density at 600 nm of 1.1) were purified according to Meisinger et al. (46), separated in a non-linear pH gradient 3–10 and stained with silver nitrate according to Bloom et al. (50). The reference map was enlarged and divided into four sections to increase the resolution. Proteins detected only in the protein pattern specific for respiratory growth are marked with an asterisk. Spots detected in less than 11 of the 12 analyzed extracts are marked with parentheses. This reference gel can be viewed at www.biochem.oulu.fi/proteomics/.

 


View this table:
[in this window]
[in a new window]
  		TABLE I
Summary of proteins identified from purified mitochondria of S. cerevisiae BJ1991 Proteins detectable only during respiratory growth are marked with an asterisk. Information about the proteins was obtained from the databases SGD, MIPS, and SWISS-PROT (SP). Subcellular localization (C, cytoplasm; M, mitochondrion (MA, matrix; IMS, intermembrane space; MIM, inner membrane; MOM, outer membrane); N, nucleus; P, peroxisome; ER, endoplasmic reticulum; LP, lipid particle; CSK, cytoskeleton; V, vacuole; R, ribosome; CW, cell wall; EX, extracellular; Me, membrane (integral [in], peripheral [pe])), according to SGD, MIPS, and SWISS-PROT (SP), taken from the literature (Exp., experimental; upper value) and predicted by TargetP (Pr., predicted, lower value) (M1–5, mitochondrial; S1–5, secretory pathway; –1–5, other; 1, high probability; 5 low probability). MTS cleavage site, according to the known mature N-terminal sequence (Exp., experimental; upper value) or calculated by TargetP (Pr., predicted; lower value). Expected spot position (pI, MM), according to the full (upper value) or mature protein sequence (lower value) calculated with Compute pI/Mw. Positions of mature sequences predicted by TargetP are marked with parentheses.

 
Subcellular Localization of the Identified Proteins—First we investigated whether the identified proteins are known as mitochondrial proteins or belong to different subcellular compartments (Table I). From the literature we found experimental evidence for a mitochondrial localization of 151 identified proteins. We also searched the databases MIPS, SGD, and SWISS-PROT, which do not strictly distinguish between experimental determined or predicted localization. In that way we confirmed the mitochondrial localization for additional eight proteins (Arg8p, Gcv2p, Lys4p, Mef1p, Mrs1p, Qcr10p, Ygl068p, and Ypr004p). For several proteins with no data available we used the bioinformatic tools PSORT II, MITOPROT, and TargetP to predict their localization. To estimate the confidence of these predictions we first processed sequences of known mitochondrial proteins. We found the highest agreement with 76.4% for TargetP (110 of 144 proteins), followed by MITOPROT and PSORT II with 70.6% and 63.9%. TargetP predicted a putative mitochondrial localization for 17 additional proteins (Ape2p, Grx5p, Rhr2p, Thr4p, Yer004p, Yer078p, Yer080p, Yer182p, Ygr086p, Yhr198p, Yil157p, Yjl200p, Ykr065p, Ylr201p, Yor108p, Ypl004p, and Ypl063p), increasing the number of mitochondrial proteins to 176.

However, for eight proteins (Hom6p, Pdh1p, Sam2p, Ydr071p, Yjr003p, Yjr085p, Ymr226p, and Ypl222p), no information about their localization was available, neither from databases nor by prediction with TargetP. Our data provide here a preliminary base to address them also as mitochondrial proteins.

A number of proteins detected in our mitochondrial fraction have been described so far for other subcellular compartments. Similar results were obtained also for mitochondrial preparations of some higher eukaryotes (27, 29, 53).

The majority (46 proteins) of the non-mitochondrial proteins identified here are cytoplasmic. According to data base information many of them are highly abundant and thus likely contaminants (Table I). Interestingly, five of them (Ach1p, Eno1p, Hxk2p, Pgk1p, and Sti1p) have been localized earlier within purified mitochondrial protein complexes (53). Among the nonmitochondrial proteins we also detected two vacuolar proteins (Tfp1p and Vma5p), three peroxisomal proteins (Fat2p, Fox1p, and Fox2p, the latter two only in the glycerol culture), a nuclear protein (Rvb1p), which was found also in other mitochondrial extracts (53), and Faa1p, which had been reported so far to be localized in lipid particles (54). Altogether, we found 54 putative contaminants in the mitochondrial extract.

However, a number of the non-mitochondrial proteins identified in the two-dimensional gel are less abundant. This argues against a contamination due to incomplete mitochondrial purification. Instead, they may indicate a functional association of the mitochondrion with other cellular compartments. This conclusion is supported by earlier studies showing specific interaction between mitochondria and ribosomes (16–18), endoplasmic reticulum (55) and cytoskeleton (29, 56). The identification of 14 endoplasmic reticulum proteins (Ayr1p, Cdc48p, Cpr5p, Cwh41p, Emp24p, Erg6p, Erg26p, Eug1p, Fpr2p, Gtt1p, Kar2p, Pdi1p, Pst2p, and Rot2p) agreed also with earlier observations (27). This lends support to the proposed interaction between endoplasmic reticulum and mitochondrion, which has been suggested to mediate the phospholipid translocation between these organelles (55). The only cytoskeleton protein detected so far in our two-dimensional gels is actin (Act1p). This could indicate a contamination due to the high abundance of actin, or, alternatively, it might originate from the interaction between cytoskeleton and mitochondrion, which has a function in positioning the organelle within the cell or mediating the transfer of mitochondria into the budding cell (29, 56).

Interestingly, several of the proteins identified here have been also found previously within different compartments (Table I). It has been shown that such "multiple localization" can be dependent on different cellular conditions, e.g. cell cycle (57) or carbon source (58). This could explain the presence of these proteins in the mitochondrial fraction and indicates a functional association with this organelle.

The subcellular localization of all proteins identified here is summarized in Fig. 2. It presents the classification into mitochondrial, "mitochondrion-associated" (endoplasmic reticulum and cytoskeleton), probable contaminants (cytoplasm, peroxisome, vacuole, nucleus, and lipid particle), and proteins of so far unknown localization.



View larger version (21K):
[in this window]
[in a new window]
  		FIG. 2.
Summary of the subcellular localization of all identified proteins. M, mitochondrion; ER, endoplasmic reticulum; CSK, cytoskeleton; C, cytoplasm; P, peroxisome; V, vacuole; N, nucleus; and LP, lipid particle.

 
Multiple Spots, Protein Modification, and Maturation—A large number of proteins were detected within multiple spots. This phenomenon has also been described before (25, 27) and indicates post-translational modification or processing. From the 459 spots identified here we found 175 single spots, whereas 195 spots appeared as "train of spots," differing only by their pI. Interestingly, 7 of all identified spots were detected at a higher molecular mass than expected, whereas 82 spots showed a decreased molecular mass.

A comparison of expected and detected spot positions within the two-dimensional gel offers the possibility to analyze modification and maturation of the protein. Hence, 18 identified proteins with known N-terminal sequences were selected as marker spots (Abf2p, Atp7p, Atp18p, Cat5p, Cox4p, Cox6p, Cox12p, Cys3p, Hem14p, Kar2p, Mas1p, Mas2p, Mrpl3p, Mrpl17p, Nde1p, Pdb1p, Pim1p, and Rim1p) to determine pI and molecular mass of all identified proteins within the two-dimensional gel (Table I). In addition, we calculated their theoretical positions based on the protein sequence with Compute pI/Mw (Table I). A comparison between theoretical and experimental data allowed new conclusions about post-translational modification, processing, or fragmentation.

A pI shift, which was found for a large number of identified spots, might indicate protein modification, e.g. acetylation and phosphorylation. N-terminal acetylation, which lowers the pI, can be excluded for mitochondrial proteins with a cleavable targeting sequence (59). Indeed, the non-mitochondrial proteins in a shifted position might be acetylated as described earlier (59). However, a more detailed characterization of post-translational modifications has to remain subject of future studies.

A lowered pI together with a slightly decreased molecular mass indicates cleavage of the mitochondrial targeting sequence (MTS) (23). All mitochondrial proteins identified here are nuclear-encoded. Thus, these proteins are commonly targeted by a specific N-terminal MTS into the organelle (16–18). To estimate whether the proteins identified here are processed upon mitochondrial import, we first calculated the expected cleavage site by using the bioinformatic tools TargetP, MITOPROT, and PSORT II. The confidence of such prediction can be estimated by processing known mature protein sequences. TargetP has been reported to have the lowest error rate (23). Our analyses revealed a correct prediction for 26 of 41 known cleavage sites. Thus, we predicted the cleavage sites for all mitochondrial proteins with TargetP and calculated the positions of unprocessed and mature proteins within the two-dimensional gel (Table I). The comparison of predicted and experimental data revealed 87 mitochondrial proteins detectable at the position of their cleaved form, including 47 mature sequences calculated by TargetP. Mcr1p was identified within two spots and might present both the unprocessed and the mature protein. Our data also suggest that 18 mitochondrial proteins are unprocessed. A significant fraction of these are well characterized proteins of the outer membrane (Fis1p, Om45p, Por1p, Por2p, Tom20p, Tom40p, and Tom70p), which could indicate that also the other unprocessed proteins, e.g. Mpm1p, Ykl027p, and Yor251p, are components of the outer membrane. At least Mpm1p has been found in mitochondrial membrane fractions before (60). For 26 gene products the corresponding pI and molecular mass shift is too small to allow discrimination to be made between uncleaved and cleaved protein in our two-dimensional gel. Interestingly, 19 proteins showed a shift indicating cleavage, but not to the position predicted for MTS processing. Moreover, for 12 proteins there is a significant shift of the spot suggesting removal of the MTS, although TargetP does not predict MTS cleavage. For 13 proteins the difference between expected and experimental position cannot be explained by MTS cleavage and might be due to additional protein modifications.

Proteins with significantly decreased molecular masses in our two-dimensional reference gel indicate protein fragmentation as described for other mitochondrial preparations (25, 27). However, in contrast to rat liver mitochondria (27) with up to 649 spots for the carbamoyl-phosphate synthase we found a maximum of nine fragments for the ketol-acid reductoisomerase Ilv5p in the yeast mitochondrial proteome.

An example for a significantly decreased molecular mass is presented by Arg5p,6p, involved in arginine biosynthesis. ARG5,6 codes for a 94.9-kDa protein, but in the two-dimensional gel it appears in multiple spots concentrated at 37 and 52 kDa. Mass spectrometry analyses of the 52-kDa spots yielded peptides spanning amino acids 90–444, whereas the 37-kDa spots corresponded to protein fragments covering amino acids 555–859. This confirms earlier studies on Arg5,6p, which is supposed to be transported as precursor into the mitochondrion and processed into Arg5p and Arg6p (51, 61). We suggest, that the 37-kDa spots present Arg6p and the 52-kDa spots Arg5p (Table I).

Abundance and Hydrophobicity—The Codon Bias Index (CBI) is indicative of the protein abundance, a CBI between 0.6 and 1 corresponds to highly abundant and a CBI near 0 to low abundant proteins (40, 62). Here, we detected 48 proteins with a CBI under 0.1. Among them are 10 proteins with a CBI below 0.01 (Mia1p, Mrp1p, Mrpl17p, Pdh1p, Rsm26p, Yhr198p, Yjr003p, Yjr100p, Ynl026p, and Yor131p) with the lowest CBI (–0.085) found for Yjr100p (Table I). Six of these low abundant proteins are mitochondrial, two are cytoplasmic, and for three the localization is still unknown.

The GRAVY (grand average of hydropathicity index) score indicates the solubility of the proteins (63). Hydrophobic proteins with a positive GRAVY value are difficult to solubilize and usually not susceptible to two-dimensional gel electrophoresis (64). Based on this value, our proteome analyses comprise 13 hydrophobic proteins (Adh1p, Atp16p, Fpr2p, Hom6p, Hsp10p, Mir1p, Oac1p, Pad1p, Pet9p, Rpp0p, Sdh4p, Yjr085p, and Ypr004p) with positive GRAVY scores up to +0.358 (Table I). These data indicate that this experimental approach does not exclude low abundant and hydrophobic proteins.

Protein Function—An overview about the functional classification of the 176 identified mitochondrial proteins according to the data base MIPS is shown in Fig. 3. A large number of proteins detected are involved in energy metabolism (13 in tricarboxylic acid cycle, 30 in respiratory chain) but also in metabolism of amino acids (24 proteins), and C-compound and carbohydrate metabolism (45 proteins). Interestingly, we detected here all known compounds of the mitochondrial pyruvate dehydrogenase complex (Lat1p, Ldp1p, Pda1p, Pdb1p, and Pdx1p). Eight identified proteins have functions in the mitochondrial protein import (Hsp60p, Mdj1p, Mge1p, Tim23p, Tim44p, Tom20p, Tom40p, and Tom70p). For 17 identified proteins no function is known at present (Ybr230p, Yer004p, Yer080p, Yer182p, Yfr011p, Ygr086p, Yhl021p, Yhr198p, Yil157p, Yjr100p, Ykl195p, Ykr065p, Ylr201p, Ynl026p, Yor215p, Ypl004p, and Ypl063p).



View larger version (23K):
[in this window]
[in a new window]
  		FIG. 3.
Functional classification of all identified mitochondrial proteins according to the MIPS data base.

 
The Yeast Mitochondrial Proteome after the Diauxic Shift— Yeast can switch from fermentative to respiratory growth after glucose exhaustion (diauxic shift), with large changes in metabolic regulation (86) and transcriptional activity (65–67). So far little information is available how this adaptation appears at the proteomic level. We forced yeast cells to pass the diauxic shift by transferring the cells from glucose to glycerol (see "Experimental Procedures" for details) and analyzed the mitochondrial proteome of seven different extracts. The protein pattern were compared with the fermentative proteome and proteins with significantly changed abundance were identified (Figs. 4 and 5 and Table II). As expected we found changes for proteins involved in the respiratory metabolism and for proteins with so far unknown function. However, the overall change in the proteome appears surprisingly small when taking into account that the diauxic shift is a dramatic change in the metabolic state and that the mitochondrion is the organelle where the adaptation to the new growth conditions mainly occurs.



View larger version (143K):
[in this window]
[in a new window]
  		FIG. 4.
Two-dimensional gel of S. cerevisiae wild type BJ1991 mitochondria characteristic for respiration. The mitochondrial proteins from a glycerol grown yeast culture were purified according to Meisinger et al. (46), separated in a non-linear pH gradient 3–10 and stained with silver nitrate according to Bloom et al. (50). Apparent pI and molecular mass values are indicated, and the selected regions for Fig. 5 are marked with boxes.

 



View larger version (115K):
[in this window]
[in a new window]
  		FIG. 5.
Comparison of protein changes in the mitochondrial proteome of S. cerevisiae wild type BJ1991 during fermentation (glucose, G) and respiration (glycerol, GLY). Selected two-dimensional gel regions are shown here enlarged, and overlapped protein spots are marked. TCA cycle, tricarboxylic acid cycle.

 


View this table:
[in this window]
[in a new window]
  		TABLE II
Summary of identified proteins influenced by the diauxic shift

Shown is the induction ({uparrow}) or repression ({downarrow}) after the diauxic shift, the transcriptional regulation (see "Experimental Procedures"), and the physiological function according to the database SGD.

 
To verify that our data were indeed from samples of cells that were glucose-repressed or had undergone the diauxic shift, we also investigated transcriptional changes parallel to changes in the proteome, using the Affymetrix system. Our results indicated that more than 4000 genes showed more than 2-fold changes in expression when comparing the transcriptional data of glucose-versus glycerol-grown cells.

The transcriptional data we obtained correlated well with previously published results (33, 35), with only minor deviations, and clearly indicated that the glucose-grown cells were glucose-repressed and that the glycerol-grown samples had undergone the diauxic shift (see data below). We focused on the subset of this data that corresponded to the 253 proteins that we were able to identify on our two-dimensional gels and 89 of them revealed more than 2-fold change (the complete data set is available at www.biochem.oulu.fi/proteomics/). At the proteomic level only 18 proteins had a significantly changed abundance (Table II), whereas the majority of proteins regulated at the transcriptional level did not show a corresponding change in the proteome. Selected marker proteins were also studied by Western blotting of the mitochondrial fraction. Por1p and Aco1p (–1.3- and +1.7-fold change in transcription after respiratory growth on glycerol) were selected as controls with constant expression. KGD1 (+3.2-fold) and SDH2 (+9.8-fold) were chosen as significantly induced genes without and with detected change at the proteomic level. In agreement with our earlier observations, only Sdh2p was detected in increased amounts in the mitochondrial proteome (Fig. 6A). It is known that mitochondria have a different morphology and enlarge after the diauxic shift (87). To estimate how much the overall mitochondrial protein mass is increased upon metabolic derepression, we analyzed the amount of Por1p and Aco1p in whole cell extracts (Fig. 6B). The result clearly shows a higher proportion of these mitochondrial markers among cellular proteins, indicating an increase in mitochondrial proteins upon metabolic derepression.



View larger version (19K):
[in this window]
[in a new window]
  		FIG. 6.
Western blot analysis of selected mitochondrial proteins. The expression of mitochondrial proteins during fermentative (glucose, G) and respiratory (glycerol, GLY) growth was analyzed by Western blot analysis using antisera directed against Aco1p, Kgd1p, Por1p, and Sdh2p. A, purified mitochondrial protein extract (10 µg/lane); B, whole cellular protein extract (5 µg/lane). The antisera were diluted 2000-fold (Aco1p), 800-fold (Kgd1p), 20.000-fold (Por1p), and 1000-fold (Sdh2p).

 
Tricarboxylic Acid Cycle—13 of the 15 known, nuclear-encoded proteins of the tricarboxylic acid cycle were identified in our two-dimensional reference gel (Aco1p, Cit1p, Idh1p, Idh2p, Kgd1p, Kgd2p, Lpd1p, Lsc1p, Lsc2p, Mdh1p, Sdh1p, Sdh2p, and Sdh4p). It is also suspected, that Yjl200p, with strong similarity to aconitate hydratases, might also have a function as Aco2p within the tricarboxylic acid cycle (68). At the transcriptional level eight tricarboxylic acid cycle compounds are more than 3-fold up- or down-regulated after glucose exhaustion (YJL200c, KGD1, KGD2, LSC2, SDH1, SDH2, SDH3, and SDH4). The protein level revealed on at least a 3-fold induction after the diauxic shift only for the compounds of the succinate dehydrogenase complex (Sdh1p, Sdh2p, and Sdh4p) (Figs. 5 and 7). Succinate dehydrogenase has a close interaction with the respiratory chain by coupling the oxidation of succinate with the transfer of electrons to ubiquinone (53). This key role for respiration might explain the strong induction after the diauxic shift, when the cellular ATP is mainly produced via the respiratory chain. The fact that the other enzymes of the tricarboxylic acid cycle are not switched off during fermentative growth (Fig. 7) can be explained by their involvement in amino acid biosynthesis, gluconeogenesis, heme biosynthesis or de novo fatty acid synthesis (68–70).



View larger version (84K):
[in this window]
[in a new window]
  		FIG. 7.
Comparison of proteins with function in tricarboxylic acid cycle during fermentation (glucose, G) and respiration (glycerol, GLY). Schematic overview about the mechanism of the tricarboxylic acid cycle according to Steinmetz et al. (15) and Przybyla-Zawislak et al. (68) (OAA, oxaloacetate; {alpha}-KG, {alpha}-ketoglutarate). Selected two-dimensional regions are shown enlarged, and overlapped protein spots are indicated. The corresponding transcriptional data (–fold change up (+) or down (–)) show the expression change after glucose exhaustion: CIT1 (+1.9), ACO1 (+1.7), YJL200c (–5.5), IDH1 (–1.1), IDH2 (–1.7), KGD1 (+3.2), KGD2 (+5.7), LPD1 (–1.4), LSC1 (+1.4), LSC2 (+3.5), SDH1 (+7.0), SDH2 (+9.8), SDH4 (+4.6), and MDH1 (+2.3).

 
Two additional proteins (Adh2p and Ald4p) were also found induced after the diauxic shift (Fig. 5). Adh2p and Ald4p catalyze a two-step conversion of ethanol to acetate under formation of NADH ("acetaldehyde pathway") (53). The induction of Adh2p and Ald4p after the diauxic shift under the current experimental conditions is not unexpected since ADH2 has been shown to be repressed by glucose (71) and indicates a derepression after glucose exhaustion. Ethanol, which is produced by fermentation of glucose and other fermentable sugars, becomes the major carbon source after the supply of these sugars has been exhausted. Thus, Adh2p and Ald4p present an alternative source of NADH, otherwise produced by the tricarboxylic acid cycle.

Respiratory Chain—Thirty proteins identified in our two-dimensional reference gel are known parts of the respiratory chain, contribute to its function (Cyb2p, Mba1p, Mcr1p, and Sco1p) or play a yet unknown role within the respiratory chain (Yor356p and Ypr004p) (Table I). Our studies revealed an at least 3-fold mRNA induction for 13 corresponding genes (ATP3, ATP5, ATP7, COR1, COX4, COX14, CYB2, MCR1, QCR2, SDH1, SDH2, SDH3, and SDH4) (see Fig. 8). As for the tricarboxylic acid cycle proteins, most changes at the transcriptional level were not reflected in the protein pattern (Fig. 8). Only Qcr7p (complex III), Cox4p (complex IV), and Cyb2p were significantly induced during respiration (Figs. 5 and 8). Remarkably, Qcr7p and Cox4p are parts of different enzyme complexes, and their up-regulation is not followed by the other members of the corresponding complexes. Cyb2p (L-lactate cytochrome c oxidoreductase) is responsible for the conversion of lactate to pyruvate in the intermembrane space. Thus, it contributes to the function of the respiratory chain by transferring electrons to cytochrome c (73), which could explain its induction.



View larger version (84K):
[in this window]
[in a new window]
  		FIG. 8.
Comparison of selected proteins with function in the respiratory chain during fermentation (glucose, G) and respiration (glycerol, GLY). Schematic overview about the mechanism of the respiratory chain according to Joseph-Horne et al. (72) and the data base MIPS (UBQ, ubiquinone:ubiquinol pool; Cyt c, cytochrome c; DHG, dehydrogenase; MA, matrix; IMS, intermembrane space; MIM, inner membrane; and MOM, outer membrane). Selected two-dimensional regions are shown enlarged, and overlapped protein spots are indicated. The corresponding transcriptional data (-fold change up (+) or down (–)) show the expression change after glucose exhaustion: NDE1 (+1.5), SDH1 (+7.0), SDH2 (+9.8), SDH4 (+4.6), ABC1 (–1.9), COR1 (+3.0), QCR2 (+4.3), QCR7 (+1.3), RIP1 (+2.5), COX4 (+3.7), COX6 (+2.0), COX12 (+2.1), COX13 (+2.8), COX14 (+3.0), ATP1 (+1.6), ATP2 (+1.7), ATP3 (+3.0), ATP4 (+2.1), ATP5 (+3.2), ATP7 (+4.3), ATP11 (+1.1), ATP16 (+2.6), CYB2 (+68.6), MBA1 (+1.5), MCR1 (+11.3), SCO1 (+1.3), and YPR004c (+1.4).

 
2-Methylcitrate Cycle—Respiratory growth on glycerol causes an induction of Pdh1p and Icl2p (Fig. 5 and Table II). At the transcriptional level both proteins were previously found to be induced by propionate and understood as a part of the 2-methylcitrate pathway (74). Because S. cerevisiae cannot grow on propionate as the sole carbon source, the physiological function of this cycle is not entirely clear (75). Apart from propionate metabolism, it may be involved in the degradation of certain amino acids via propionyl-CoA and also connected to the {beta}-oxidation of odd-chain fatty acids (76, 77). The Escherichia coli PrpD, homolog to Pdh1p, has a 2-methylcitrate dehydratase activity and can also use citrate and isocitrate as substrates (78). Thus, Pdh1p might take part in the conversion of 2-methylcitrate to 2-methylisocitrate. ICL2 encodes a 2-methylisocitrate lyase, which catalyzes the consecutive cleavage of 2-methylisocitrate to pyruvate and succinate (76). Both proteins might be responsible for the generation of succinate and pyruvate by metabolizing threonine as an additional source of succinate. This assumption is supported by the fact that the corresponding enzyme within the tricarboxylic acid cycle (succinate dehydrogenase) is also up-regulated (see above).

Peroxisomal {beta}-Oxidation, Acetyl-CoA, and NADH—Three peroxisomal proteins (Fat2p, Fox1p, and Fox2p) were induced (Fat2p) or appeared additionally (Fox1p and Fox2p) in the mitochondrial proteome after the diauxic shift. Fox1p and Fox2p have functions in the peroxisomal {beta}-oxidation of fatty acids, but the role of Fat2p is still unknown. Peroxisomal proteins have been shown earlier to be repressed by glucose (71). The presence of Fat2p, Fox1p, and Fox2p in our two-dimensional gels indicates this derepression after glucose exhaustion.

The end products of this {beta}-oxidation, acetyl-CoA, and NADH generated inside the peroxisome must be transported into the mitochondrion for ATP generation and biosynthetic processes (79). Here we found Ach1p and Cat2p also induced under respiratory growth. Cat2p catalyzes the transferring of an acetyl-group from acetyl-CoA to carnitine, which can be transported across membranes to the mitochondrion (80). Ach1p, an acetyl-CoA hydrolase, is known to be induced after glucose limitation (32) or addition of ethanol (34). This suggests a role for Ach1p in regulating the intracellular acetyl-CoA pool.

Ybr230p and Om45p—Om45p and Ybr230p are also induced by growth on glycerol (Fig. 5). The function of both proteins is unknown so far, but the transcriptional induction of the corresponding genes after glucose limitation (Table II) suggests a role in the respiratory metabolism. Om45p has been earlier identified in a purified mitochondrial enzyme complex together with components of the tricarboxylic acid cycle and respiratory chain (53). Likewise Ybr230p has been suggested to be co-regulated with proteins from tricarboxylic acid cycle and respiratory chain (81). On that background our results indicate a connection of both enzymes to tricarboxylic acid cycle and respiratory chain. Om45p, a mitochondrial outer membrane protein, has been suggested to function in protein import (82). Based on our results and the known localization, we propose a role for Om45p in the import of metabolic intermediates or proteins required for respiratory growth.

The proteomic study presented here reveals that the yeast mitochondrial proteome is kept in a steady-state even upon large metabolic changes. Thus, the majority of proteins involved in respiratory metabolism is present also during fermentative growth, probably with lower activity or in different functional context. Tricarboxylic acid cycle enzymes for instance are also involved in different synthetic pathways (83). This could explain why the corresponding proteins are present under both conditions. The fact, that only some components of the respiratory chain are significantly up-regulated may indicate key functions in complex assembly or electron transfer under these conditions.

Our analyses and previous reports on transcriptional regulation (33, 35–37), however, revealed transcriptional changes for a much larger number of genes than found here at the proteome level. Partially, the transcriptional up-regulation can be accounted for by an overall increase in mitochondria upon the diauxic shift. However, the changes in transcriptional activity are heterogenous and do not reflect the situation found for the proteome. First, the discrepancy between transcriptome and proteome can simply reflect differences in translational efficiency for individual proteins. In addition, we assume that this discrepancy reflects differences in protein turnover for individual proteins. The turnover rate is likely to change under different physiological conditions, and the shift from fermentation to respiratory metabolism has probably pronounced effect on the "aging" of individual proteins.


   FOOTNOTES
 
* This work was supported by grants from the Academy of Finland and Sigrid Jusélius Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed. Tel.: 358-8-553-1209; Fax: 358-8-553-1141; E-mail: Steffen.Ohlmeier{at}oulu.fi.

1 The abbreviations used are: MTS, mitochondrial targeting sequence; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; CBI, Codon bias index; GRAVY, grand average of hydropathicity index. Back


   ACKNOWLEDGMENTS
 
We thank Eeva-Liisa Stefanius and Marika Kamps for excellent technical assistance and Jussi Vuoristo for the Affymetrix services. We are grateful to Alexander Tzagoloff, Benedikt Westermann, and Bernard D. Lemire for the gift of antibodies.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 

  1. Fossati, G., Moulding, D. A., Spiller, D. G., Moots, R. J., White, M. R., and Edwards, S. W. (2003) J. Immunol. 170, 1964–1972[Abstract/Free Full Text]
  2. La Piana, G., Marzulli, D., Consalvo, M. I., and Lofrumento, N. E. (2003) Arch. Biochem. Biophys. 410, 201–211[CrossRef][Medline] [Order article via Infotrieve]
  3. Jambrina, E., Alonso, R., Alcalde, M., Rodriguez, M. M., Serrano, A., Martinez-A., C., Garcia-Sancho, J., and Izquierdo, M. (2003) J. Biol. Chem. 278, 14134–14145[Abstract/Free Full Text]
  4. Bota, D. A., and Davies, K. J. A. (2001) Mitochondrion 1, 33–49[CrossRef][Medline] [Order article via Infotrieve]
  5. Attardi, G. (2002) Mitochondrion 2, 27–37[CrossRef][Medline] [Order article via Infotrieve]
  6. Sun, J., Folk, D., Bradley, T. J., and Tower, J. (2002) Genetics 161, 661–672[Abstract/Free Full Text]
  7. Swerdlow, R. H., and Kish, S. J. (2002) Int. Rev. Neurobiol. 53, 341–385[Medline] [Order article via Infotrieve]
  8. Sherer, T. B., Betarbet, R., and Greenamyre, J. T. (2002) Neuroscientist 8, 192–197[Abstract/Free Full Text]
  9. Maassen, J. A. (2002) Am. J. Med. Genet. 115, 66–70[CrossRef][Medline] [Order article via Infotrieve]
  10. De Marcos Lousa, C., Trezeguet, V., Dianoux, A. C., Brandolin, G., and Lauquin, G. J. (2002) Biochemistry 41, 14412–14420[CrossRef][Medline] [Order article via Infotrieve]
  11. Rabilloud, T., Strub, J. M., Carte, N., Luche, S., Van Dorsselaer, A., Lunardi, J., Giege, R., and Florentz, C. (2002) Biochemistry 41, 144–150[CrossRef][Medline] [Order article via Infotrieve]
  12. Chinnery, P. F. (2002) Mitochondrion 2, 149–155[CrossRef][Medline] [Order article via Infotrieve]
  13. Pandolfo, M. (2002) Blood Cells Mol. Dis. 29, 536–547[CrossRef][Medline] [Order article via Infotrieve]
  14. Tomlinson, I. P., Alam, N. A., Rowan, A. J., Barclay, E., Jaeger, E. E., Kelsell, D., Leigh, I., Gorman, P., Lamlum, H., Rahman, S., Roylance, R. R., Olpin, S., Bevan, S., Barker, K., Hearle, N., Houlston, R. S., Kiuru, M., Lehtonen, R., Karhu, A., Vilkki, S., Laiho, P., Eklund, C., Vierimaa, O., Aittomaki, K., Hietala, M., Sistonen, P., Paetau, A., Salovaara, R., Herva, R., Launonen, V., and Aaltonen, L. A. (2002) Nat. Genet. 30, 406–410[CrossRef][Medline] [Order article via Infotrieve]
  15. Steinmetz, L. M., Scharfe, C., Deutschbauer, A. M., Mokranjac, D., Herman, Z. S., Jones, T., Chu, A. M., Giaever, G., Prokisch, H., Oefner, P. J., and Davis, R. W. (2002) Nat. Genet. 31, 400–404[Medline] [Order article via Infotrieve]
  16. Marc, P., Margeot, A., Devaux, F., Blugeon, C., Corral-Debrinski, M., and Jacq, C. (2002) EMBO Rep. 3, 159–164[CrossRef][Medline] [Order article via Infotrieve]
  17. Nelson, R. J., Ziegelhoffer, T., Nicolet, C., Werner-Washburne, M., and Craig, E. A. (1992) Cell 71, 97–105[CrossRef][Medline] [Order article via Infotrieve]
  18. Lithgow, T. (2000) FEBS Lett. 476, 22–26[CrossRef][Medline] [Order article via Infotrieve]
  19. Karlberg, O., Canbäck, B., Kurland, C. G., and Andersson, S. G. E. (2000) Yeast 17, 170–187[CrossRef][Medline] [Order article via Infotrieve]
  20. Kumar, A., Agarwal, S., Heyman, J. A., Matson, S., Heidtman, M., Piccirillo, S., Umansky, L., Drawid, A., Jansen, R., Liu, Y., Cheung, K. H., Miller, P., Gerstein, M., Roeder, G. S., and Snyder, M. (2002) Genes Dev. 16, 707–719[Abstract/Free Full Text]
  21. Scheffler, I. E. (2001) Mitochondrion 1, 3–31[CrossRef][Medline] [Order article via Infotrieve]
  22. Kruft, V., Eubel, H., Jänsch, L., Werhahn, W., and Braun, H.-P. (2001) Plant Physiol. 127, 1694–1710[Abstract/Free Full Text]
  23. Millar, A. H., Sweetlove, L. J., Giegé, P., and Leaver, C. J. (2001) Plant Physiol. 127, 1711–1727[Abstract/Free Full Text]
  24. Werhahn, W., and Braun, H. P. (2002) Electrophoresis 23, 640–646[CrossRef][Medline] [Order article via Infotrieve]
  25. Bardel, J., Louwagie, M., Jaquinod, M., Jourdain, A., Luche, S., Rabilloud, T., Macherel, D., Garin, J., and Bourguignon, J. (2002) Proteomics 2, 880–898[CrossRef][Medline] [Order article via Infotrieve]
  26. Kernec, F., Unlu, M., Labeikovsky, W., Minden, J. S., and Koretsky, A. P. (2001) Physiol. Genomics 6, 117–128[Abstract/Free Full Text]
  27. Fountoulakis, M., Berndt, P., Langen, H., and Suter, L. (2002) Electrophoresis 23, 311–328[CrossRef][Medline] [Order article via Infotrieve]
  28. Brookes, P. S., Pinner, A., Ramachandran, A., Coward, L., Barnes, S., Kim, H., and Darley-Usmar, V. M. (2002) Proteomics 2, 969–977[CrossRef][Medline] [Order article via Infotrieve]
  29. Rabilloud, T., Kieffer, S., Procaccio, V., Louwagie, M., Courchesne, P. L., Patterson, S. D., Martinez, P., Garin, J., and Lunardi, J. (1998) Electrophoresis 19, 1006–1014[CrossRef][Medline] [Order article via Infotrieve]
  30. Lopez, M. F., Kristal, B. S., Chernokalskaya, E., Lazarev, A., Shestopalov, A. I., Bogdanova, A., and Robinson, M. (2000) Electrophoresis 21, 3427–3440[CrossRef][Medline] [Order article via Infotrieve]
  31. Entian, K. D., and Barnett, J. A. (1992) Trends Biochem. Sci. 17, 506–510[CrossRef][Medline] [Order article via Infotrieve]
  32. Boy-Marcotte, E., Perrot, M., Bussereau, F., Boucherie, H., and Jacquet, M. (1998) J. Bacteriol. 180, 1044–1052[Abstract/Free Full Text]
  33. DeRisi, J. L., Iyer, V. R., and Brown, P. O. (1997) Science 278, 680–686[Abstract/Free Full Text]
  34. Haurie, V., Perrot, M., Mini, T., Jeno, P., Sagliocco, F., and Boucherie, H. (2001) J. Biol. Chem. 276, 76–85[Abstract/Free Full Text]
  35. Kal, A. J., van Zonneveld, A. J., Benes, V., van den Berg, M., Koerkamp, M. G., Albermann, K., Strack, N., Ruijter, J. M., Richter, A., Dujon, B., Ansorge, W., and Tabak, H. F. (1999) Mol. Biol. Cell 10, 1859–1872[Abstract/Free Full Text]
  36. Karpichev, I. V., Small, G. M. (1998) Mol. Cell Biol. 18, 6560–6570[Abstract/Free Full Text]
  37. Hardwick, J. S., Kuruvilla, F. G., Tong, J. K., Shamji, A. F., and Schreiber, S. L. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 14866–14870[Abstract/Free Full Text]
  38. Blom, J., De Mattos, M. J., and Grivell, L. A. (2000) Appl. Environ. Microbiol. 66, 1970–1973[Abstract/Free Full Text]
  39. Igual, J. C., and Navarro, B. (1996) Mol. Gen. Genet. 252, 446–455[Medline] [Order article via Infotrieve]
  40. Boucherie, H., Sagliocco, F., Joubert, R., Maillet, I., Labarre, J., and Perrot, M. (1996) Electrophoresis 17, 1683–1699[CrossRef][Medline] [Order article via Infotrieve]
  41. Griffin, T. J., Gygi, S. P., Ideker, T., Rist, B., Eng, J., Hood, L., and Aebersold, R. (2002) Mol. Cell Proteomics 1, 323–333[Abstract/Free Full Text]
  42. Gygi, S. P., Rochon, Y., Franza, B. R., and Aebersold, R. (1999) Mol. Cell Biol. 19, 1720–1730[Abstract/Free Full Text]
  43. Anderson, L., and Seilhamer, J. (1997) Electrophoresis 18, 533–537[CrossRef][Medline] [Order article via Infotrieve]
  44. Futcher, B., Latter, G. I., Monardo, P., McLaughlin, C. S., and Garrels, J. I. (1999) Mol. Cell Biol. 19, 7357–7368[Abstract/Free Full Text]
  45. Jones, E. W. (1977) Genetics 85, 23–33[Abstract/Free Full Text]
  46. Meisinger, C., Sommer, T., and Pfanner, N. (2000) Anal. Biochem. 287, 339–342[CrossRef][Medline] [Order article via Infotrieve]
  47. Bradford, M. M. (1976) Anal. Biochem. 72, 248–254[CrossRef][Medline] [Order article via Infotrieve]
  48. Buttner, K., Bernhardt, J., Scharf, C., Schmid, R., Mader, U., Eymann, C., Antelmann, H., Volker, A., Volker, U., and Hecker, M. (2001) Electrophoresis 22, 2908–2935[CrossRef][Medline] [Order article via Infotrieve]
  49. Görg, A., Boguth, G., Obermaier, C., Posch, A., and Weiss, W. (1995) Electrophoresis 16, 1079–1086[CrossRef][Medline] [Order article via Infotrieve]
  50. Bloom, H., Beier, H., and Gross, H. (1987) Electrophoresis 8, 93–99[CrossRef]
  51. Boonchird, C., Messenguy, F., and Dubois, E. (1991) Eur. J. Biochem. 199, 325–335[Medline] [Order article via Infotrieve]
  52. Liu, Y., Van Heeswijck, R., Hoj, P., and Hoogenraad, N. (1995) Eur. J. Biochem. 228, 291–296[Medline] [Order article via Infotrieve]
  53. Grandier-Vazeille, X., Bathany, K., Chaignepain, S., Camougrand, N., Manon, S., and Schmitter, J. M. (2001) Biochemistry 40, 9758–9769[CrossRef][Medline] [Order article via Infotrieve]
  54. Athenstaedt, K., Zweytick, D., Jandrositz, A., Kohlwein, S. D., and Daum, G. (1999) J. Bacteriol. 181, 6441–6448[Abstract/Free Full Text]
  55. Achleitner, G., Gaigg, B., Krasser, A., Kainersdorfer, E., Kohlwein, S. D., Perktold, A., Zellnig, G., and Daum, G. (1999) Eur. J. Biochem. 264, 545–553[Medline] [Order article via Infotrieve]
  56. Hermann, G. J., King, E. J., and Shaw, J. M. (1997) J. Cell Biol. 137, 141–153[Abstract/Free Full Text]
  57. Madeo, F., Schlauer, J., Zischka, H., Mecke, D., and Frohlich, K. U. (1998) Mol. Biol. Cell 9, 131–141[Abstract/Free Full Text]
  58. Farcasanu, I. C., Hirata, D., Tsuchiya, E., Mizuta, K., and Miyakawa, T. (1999) Biosci. Biotechnol. Biochem. 63, 1871–1881[CrossRef][Medline] [Order article via Infotrieve]
  59. Polevoda, B., Norbeck, J., Takakura, H., Blomberg, A., and Sherman, F. (1999) EMBO J. 18, 6155–6168[CrossRef][Medline] [Order article via Infotrieve]
  60. Inadome, H., Noda, Y., Adachi, H., and Yoda, K. (2001) Biosci. Biotechnol. Biochem. 65, 2577–2580[CrossRef][Medline] [Order article via Infotrieve]
  61. Abadjieva, A., Pauwels, K., Hilven, P., and Crabeel, M. (2001) J. Biol. Chem. 276, 42869–42880[Abstract/Free Full Text]
  62. Bennetzen, J. L., and Hall, B. D. (1982) J. Biol. Chem. 257, 3026–3031[Abstract/Free Full Text]
  63. Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 157, 105–132[CrossRef][Medline] [Order article via Infotrieve]
  64. Herbert, B. (1999) Electrophoresis 20, 660–663[CrossRef][Medline] [Order article via Infotrieve]
  65. Gancedo, C., and Serrano, R. (1989) in The Yeast (Harrison, J. S., and Rose, A. H., eds) pp. 205–259, Academic Press, London
  66. Ronne, H. (1995) Trends Genet. 11, 12–17[CrossRef][Medline] [Order article via Infotrieve]
  67. De Winde, J. H., Thevelein, J. M., and Windercrix, J. (1997) in Yeast Stress Responses (Hohmann, S., and Mager, W. H., eds) pp. 7–52, Springer, Berlin
  68. Przybyla-Zawislak, B., Gadde, D. M., Ducharme, K., and McCammon, M. T. (1999) Genetics 152, 153–166[Abstract/Free Full Text]
  69. Maaheimo, H., Fiaux, J., Cakar, Z. P., Bailey, J. E., Sauer, U., and Szyperski, T. (2001) Eur. J. Biochem. 268, 2464–2479[Medline] [Order article via Infotrieve]
  70. McCammon, M. T., Epstein, C. B., Przybyla-Zawislak, B., McAlister-Henn, L., and Butow, R. A. (2003) Mol. Biol. Cell 14, 958–972[Abstract/Free Full Text]
  71. Gancedo, J. M. (1998) Microbiol. Mol. Biol. Rev. 62, 334–361[Abstract/Free Full Text]
  72. Joseph-Horne, T., Hollomon, D. W., and Wood, P. M. (2001) Biochim. Biophys. Acta 1504, 179–195[Medline] [Order article via Infotrieve]
  73. Capeillere-Blandin, C. (1982) Eur. J. Biochem. 128, 533–542[Medline] [Order article via Infotrieve]
  74. Epstein, C. B., Waddle, J. A., Hale, W., IV, Dave, V., Thornton, J., Macatee, T. L., Garner, H. R., and Butow, R. A. (2001) Mol. Biol. Cell 12, 297–308[Abstract/Free Full Text]
  75. Pronk, J. T., van der Linden-Beuman, A., Verduyn, C., Scheffers, W. A., and van Dijken, J. P. (1994) Microbiology 140, 717–722[Abstract/Free Full Text]
  76. Luttik, M. A., Overkamp, K. M., Kotter, P., de Vries, S., van Dijken, J. P., and Pronk, J. T. (1998) J. Biol. Chem. 273, 24529–24534[Abstract/Free Full Text]
  77. Metzler, D. E. (1977) Biochemistry: the Chemical Reactions of Living Cells, pp. 100–104, Academic Press, Inc., New York
  78. Blank, L., Green, J., and Guest, J. R. (2002) Microbiology 148, 133–146[Abstract/Free Full Text]
  79. Elgersma, Y., and Tabak, H. F. (1996) Biochim. Biophys. Acta 1286, 269–283[Medline] [Order article via Infotrieve]
  80. Van Roermund, C. W., Hettema, E. H., van den Berg, M., Tabak, H. F., and Wanders, R. J. (1999) EMBO J. 18, 5843–5852[CrossRef][Medline] [Order article via Infotrieve]
  81. Lascaris, R., Bussemaker, H. J., Boorsma, A., Piper, M., Van Der Spek, H., Grivell, L., and Blom, J. (2003) Genome. Biol. 4, R3[CrossRef][Medline] [Order article via Infotrieve]
  82. Yaffe, M. P., Jensen, R. E., and Guido E. C. (1989) J. Biol. Chem. 264, 21091–21096[Abstract/Free Full Text]
  83. McAlister-Henn, L., and Small, W. C. (1997) Prog. Nucleic Acids Res. Mol. Biol. 57, 317–339[Medline] [Order article via Infotrieve]
  84. Ausubel, F. M., Brent, R. E., Kingston, D. D., Moore, J. G., and Seidman, J. A. (eds) (1989) Current Protocols in Molecular Biology, 13.12.1, John Wiley and Sons, Inc., New York
  85. Miinalainen, I. J., Chen, Z. J., Torkko, J. M., Pirila, P. L., Sormunen, R. T., Bergmann, U., Qin, Y. M., and Hiltunen, J. K. (2003) J. Biol. Chem. 278, 20154–20161[Abstract/Free Full Text]
  86. Perlman, P. S., and Mahler, H. R. (1974) Arch. Biochem. Biophys. 162, 248–271[CrossRef][Medline] [Order article via Infotrieve]
  87. Egner, A., Jakobs, S., and Hell, S. W. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 3370–3375[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Mol. Cell. ProteomicsHome page
A. C. Smith and A. J. Robinson
MitoMiner, an Integrated Database for the Storage and Analysis of Mitochondrial Proteomics Data
Mol. Cell. Proteomics, June 1, 2009; 8(6): 1324 - 1337.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
G. R. Stuart, W. C. Copeland, and M. K. Strand
Construction and application of a protein and genetic interaction network (yeast interactome)
Nucleic Acids Res., April 1, 2009; 37(7): e54 - e54.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
U. Gey, C. Czupalla, B. Hoflack, G. Rodel, and U. Krause-Buchholz
Yeast Pyruvate Dehydrogenase Complex Is Regulated by a Concerted Activity of Two Kinases and Two Phosphatases
J. Biol. Chem., April 11, 2008; 283(15): 9759 - 9767.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. ProteomicsHome page
J. Reinders, K. Wagner, R. P. Zahedi, D. Stojanovski, B. Eyrich, M. van der Laan, P. Rehling, A. Sickmann, N. Pfanner, and C. Meisinger
Profiling Phosphoproteins of Yeast Mitochondria Reveals a Role of Phosphorylation in Assembly of the ATP Synthase
Mol. Cell. Proteomics, November 1, 2007; 6(11): 1896 - 1906.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Sci.Home page
B. Almeida, S. Buttner, S. Ohlmeier, A. Silva, A. Mesquita, B. Sampaio-Marques, N. S. Osorio, A. Kollau, B. Mayer, C. Leao, et al.
NO-mediated apoptosis in yeast
J. Cell Sci., September 15, 2007; 120(18): 3279 - 3288.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Cell Mol. Bio.Home page
M. Santos, R. S. Moura, S. Gonzaga, C. Nogueira-Silva, S. Ohlmeier, and J. Correia-Pinto
Embryonic Essential Myosin Light Chain Regulates Fetal Lung Development in Rats
Am. J. Respir. Cell Mol. Biol., September 1, 2007; 37(3): 330 - 338.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
Q. Zhong, G. Li, J. Gvozdenovic-Jeremic, and M. L. Greenberg
Up-regulation of the Cell Integrity Pathway in Saccharomyces cerevisiae Suppresses Temperature Sensitivity of the pgs1{Delta} Mutant
J. Biol. Chem., June 1, 2007; 282(22): 15946 - 15953.
[Abstract] [Full Text] [PDF]

Home page
 Mol Biol EvolHome page
I. E. Gentle, A. J. Perry, F. H. Alcock, V. A. Likic, P. Dolezal, E. T. Ng, A. W. Purcell, M. McConnville, T. Naderer, A.-L. Chanez, et al.
Conserved Motifs Reveal Details of Ancestry and Structure in the Small TIM Chaperones of the Mitochondrial Intermembrane Space
Mol. Biol. Evol., May 1, 2007; 24(5): 1149 - 1160.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
V. R. Dasari, H. K. Anandatheerthavarada, M.-A. Robin, E. Boopathi, G. Biswas, J.-K. Fang, D. W. Nebert, and N. G. Avadhani
Role of Protein Kinase C-mediated Protein Phosphorylation in Mitochondrial Translocation of Mouse CYP1A1, Which Contains a Non-canonical Targeting Signal
J. Biol. Chem., October 13, 2006; 281(41): 30834 - 30847.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Biol. CellHome page
R. P. Zahedi, A. Sickmann, A. M. Boehm, C. Winkler, N. Zufall, B. Schonfisch, B. Guiard, N. Pfanner, and C. Meisinger
Proteomic Analysis of the Yeast Mitochondrial Outer Membrane Reveals Accumulation of a Subclass of Preproteins
Mol. Biol. Cell, March 1, 2006; 17(3): 1436 - 1450.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
T. Major, B. von Janowsky, T. Ruppert, A. Mogk, and W. Voos
Proteomic Analysis of Mitochondrial Protein Turnover: Identification of Novel Substrate Proteins of the Matrix Protease Pim1
Mol. Cell. Biol., February 1, 2006; 26(3): 762 - 776.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
H. Prokisch, C. Andreoli, U. Ahting, K. Heiss, A. Ruepp, C. Scharfe, and T. Meitinger
MitoP2: the mitochondrial proteome database--now including mouse data
Nucleic Acids Res., January 1, 2006; 34(suppl_1): D705 - D711.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
T. Kimura, Y. Hosoda, Y. Sato, Y. Kitamura, T. Ikeda, T. Horibe, and M. Kikuchi
Interactions among Yeast Protein-Disulfide Isomerase Proteins and Endoplasmic Reticulum Chaperone Proteins Influence Their Activities
J. Biol. Chem., September 9, 2005; 280(36): 31438 - 31441.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Biol. CellHome page
M. J. Brauer, A. J. Saldanha, K. Dolinski, and D. Botstein
Homeostatic Adjustment and Metabolic Remodeling in Glucose-limited Yeast Cultures
Mol. Biol. Cell, May 1, 2005; 16(5): 2503 - 2517.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. ProteomicsHome page
K. Rezaul, L. Wu, V. Mayya, S.-I. Hwang, and D. Han
A Systematic Characterization of Mitochondrial Proteome from Human T Leukemia Cells
Mol. Cell. Proteomics, February 1, 2005; 4(2): 169 - 181.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. Augustin, M. Nolden, S. Muller, O. Hardt, I. Arnold, and T. Langer
Characterization of Peptides Released from Mitochondria: EVIDENCE FOR CONSTANT PROTEOLYSIS AND PEPTIDE EFFLUX
J. Biol. Chem., January 28, 2005; 280(4): 2691 - 2699.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. L. Tai, V. M. Boer, P. Daran-Lapujade, M. C. Walsh, J. H. de Winde, J.-M. Daran, and J. T. Pronk
Two-dimensional Transcriptome Analysis in Chemostat Cultures: COMBINATORIAL EFFECTS OF OXYGEN AVAILABILITY AND MACRONUTRIENT LIMITATION IN SACCHAROMYCES CEREVISIAE
J. Biol. Chem., January 7, 2005; 280(1): 437 - 447.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. ProteomicsHome page
X.-S. Jiang, J. Dai, Q.-H. Sheng, L. Zhang, Q.-C. Xia, J.-R. Wu, and R. Zeng
A Comparative Proteomic Strategy for Subcellular Proteome Research: Icat Approach Coupled with Bioinformatics Prediction to Ascertain Rat Liver Mitochondrial Proteins and Indication of Mitochondrial Localization for Catalase
Mol. Cell. Proteomics, January 1, 2005; 4(1): 12 - 34.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Biol. CellHome page
G. G. Perrone, C. M. Grant, and I. W. Dawes
Genetic and Environmental Factors Influencing Glutathione Homeostasis in Saccharomyces cerevisiae
Mol. Biol. Cell, January 1, 2005; 16(1): 218 - 230.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Biol. CellHome page
S. Aoki, S. Toda, T. Ando, and H. Sugihara
Bone Marrow Stromal Cells, Preadipocytes, and Dermal Fibroblasts Promote Epidermal Regeneration in Their Distinctive Fashions
Mol. Biol. Cell, October 1, 2004; 15(10): 4647 - 4657.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
P. A. Cobine, L. D. Ojeda, K. M. Rigby, and D. R. Winge
Yeast Contain a Non-proteinaceous Pool of Copper in the Mitochondrial Matrix
J. Biol. Chem., April 2, 2004; 279(14): 14447 - 14455.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/6/3956    most recent
M310160200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ohlmeier, S.
Right arrow Articles by Bergmann, U.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ohlmeier, S.
Right arrow Articles by Bergmann, U.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2004 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement