journal of vol. 264, no. 7, of 5, pp. 1989 by for …vol. 264, no. 7, issue of march 5, pp....

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology. h C .

Vol. 264, No. 7, Issue of March 5, pp. 3723-3731,1989 Printed in U. S. A.

Mutational Analysis of the Mitochondrial Rieske Iron-Sulfur Protein of Saccharomyces cerevisiae 11. BIOCHEMICAL CHARACTERIZATION OF TEMPERATURE-SENSITIVE RIP1- MUTATIONS*

(Received for publication, May 24, 1988)

Per 0. Ljungdahlz, Joe D. Beckmann6, and Bernard L. Trumpower From the Department of Biochemistry, Dartmouth Medical School, Hanouer, New Hampshire 03756

Although the function of the Rieske iron-sulfur protein is generally understood, little is known of how the structure of this protein supports its mechanistic role in electron transfer in the cytochrome b c ~ complex. TO better understand the structural basis of iron-sulfur protein function, we have undertaken a mutational analysis of the gene encoding this protein and initially isolated five temperature-sensitive iron-sulfur protein mutants (Beckmann, J. D., Ljungdahl, P. O., and Trumpower, B. L. (1989) J. Biol. Chern. 264, 3713- 3722).

Each of the five ts-ripl- mutants exhibited pleiotropic effects. Although the mutant iron-sulfur proteins manifest several in vitro phenotypes in common, each exhibited unique characteristics. All of the ts- ripl- mutations resulted in membranes with decreased ubiquinol-cytochrome c oxidoreductase activities and decreased thermostability compared to membranes containing wild type iron-sulfur protein. All of the mutations conferred slight but significant resistance to the respiratory inhibitor myxothiazol, and one mutant was hypersensitive to inhibition by UHDBT, a structural analog of ubiquinone. In addition, one of the mutations completely blocks post-translational processing of the iron-sulfur protein, leading to accumulation of pre-iron-sulfur protein in mitochondrial membranes at nonpermissive temperatures.

Finally, a mutation 12-amino acid residues away from the carboxyl terminus (2035) results in an extremely unstable protein. This region of the protein may be essential in blocking degradation of pre-iron- sulfur protein by cytoplasmic proteases as the protein is imported into the mitochondria, or may be a “degradation signal,” which tags the iron-sulfur protein for turnover.

Primary amino acid sequences have been determined for Rieske iron-sulfur proteins from a variety of photosynthetic and nonphotosynthetic organisms (1-10). These sequences have revealed a high degree of similarity, especially in the carboxyl-terminal region of the protein. The iron-sulfur protein of Saccharomyces cerevisiae is encoded by a nuclear gene (RIP1) and is imported into the mitochondria. This post-

GM 20379 and Public Health Service Fellowship GM 10575-02 (to * This work was supported by National Institutes of Health Grant

J. D. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Cambridge Center, Cambridge, MA 02142. $ Present address: Whitehead Institute for Biomedical Research, 9

son 2051, Omaha, NE 68105. Present address: University of Nebraska Medical School, Swan-

translational process results in the cleavage of a 30-amino acid leader sequence (8,11,12). It appears that the iron-sulfur protein follows a maturation process similar to other mitochondrial proteins containing prosthetic groups (13-15).

The mitochondrial import and subsequent processing reactions of the Neurospora crassa Rieske iron-sulfur protein have been well characterized (16), and a two-step processing pathway has been postulated. Whether a similar processing pathway exists in yeast is not known. In particular, it is not established if the yeast pre-iron-sulfur protein is proteolytically cleaved to mature-iron-sulfur protein in one (11) or two steps (12).

Electron flow through the cytochrome bcl complex occurs by a protonmotive Q-cycle (17-20), which includes two path- ways of cytochrome b reduction. The first pathway, which is thermodynamically favored, is sensitive to myxothiazol inhibition (21). Cytochrome b reduction through this pathway is linked to reduction of iron-sulfur protein, which generates an unstable, low potential ubisemiquinone anion at a reaction site localized to the electropositive side of the membrane (“center P”). This semiquinone reduces the b cytochromes through the low potential b-566. Reduced cytochrome b is then reoxidized by ubiquinone or ubisemiquinone anion in an antimycin-sensitive reaction at a site localized to the electro- negative side of the membrane (“center N”). If production of ubisemiquinone anion at center P is blocked, either by myxothiazol or because iron-sulfur protein is reduced, cytochrome b can be reduced under pre-steady state conditions by reversal of the antimycin-sensitive pathway at center N. UHDBT,’ a structural analog of ubiquinone, blocks electron transfer by binding to reduced iron-sulfur protein, thus preventing production of ubisemiquinone anion at center P and reduction of cytochrome CI.

Antimycin, myxothiazol, and UHDBT disrupt electron transfer reactions involving ubiquinone/ubiquinol at either center N or center P. Antimycin is characteristic of inhibitors which bind to a site at center N on cytochrome b (22). Center N is distal to the ubiquinol oxidation site center P, which is most likely formed by interaction between the iron-sulfur protein and cytochrome b.

There are two classes of center P inhibitors, both of which mimic removal of the iron-sulfur protein from the cytochrome bcl complex (23). Myxothiazol is an example of the first class, which bind to cytochrome b at an interface between cytochrome b and the iron-sulfur protein (24). UHDBT is repre- sentative of the second class of center P inhibitors, which apparently bind to the iron-sulfur protein (25).

The iron-sulfur protein must perform several functions if

The abbreviations used are: UHDBT, 5-N-undecyl-6-hydroxy- 4,7-dioxobenzothiazole; SDS, sodium dodecylsulfate; MES, &mor- pholineethanesulfonic acid.

3723

3724 Temperature-sensitive Iron-Sulfur Protein Mutants

the protonmotive Q-cycle correctly describes electron flow through the cytochrome bcl complex. The iron-sulfur protein catalyzed reaction QH2 + ISP,, + Q; + ISPred + 2H+ is the thermodynamic driving reaction and the energy-transducing reaction in which protons are released to the cytoplasm (19). Therefore, the iron-sulfur protein must have a quinol-binding site or form such a site with polypeptide domains of cytochrome b. The iron-sulfur protein, either alone or in concert with cytochrome 6 , must prevent the intermediate semiquinone anion from diffusing away and must direct its reactivity to ensure that the semiquinone intermediate reduces cytochrome b. Reduced iron-sulfur protein must also transfer an electron to cytochrome cl. Finally, the reaction catalyzed by the iron-sulfur protein at center P is highly pH dependent (A& = 120 mV pH"), and may therefore be a site of respiratory control (20).

Much is known about the reactions that the iron-sulfur protein catalyzes, but little is known of how the structure of this protein fulfills its mechanistic role in the Q-cycle. To elucidate the structural basis of iron-sulfur protein function, we have initiated a mutational analysis of the gene encoding this protein. Five temperature-sensitive mutations within the iron-sulfur protein gene that affect its stability and electron transfer function have been isolated. This report describes the biochemical characterization of mitochondrial membranes carrying mutant forms of the Rieske iron-sulfur protein.

EXPERIMENTAL PROCEDURES

Materials-Serum albumin (essentially fatty acid free), horse heart cytochrome c, Trizma base, EDTA, diisopropylfluorophosphate, phenylmethylsulfonyl fluoride, antimycin, 4-chloro-1-naphthol, Tri- ton X-100, Ponceau S, adenine sulfate, and lyticase were obtained from Sigma. SDS was from Rio-Rad. Myxothlazol was from Boehrin- ger Mannheim. Zymolyase-100T was obtained from Miles Scientific. Peroxidase-conjugated anti-mouse immunoglobulins were obtained from Cooper Biomedical Inc. Yeast strains are listed in Table I of the preceding paper.

Yeast Culture Conditions-Commonly used media are described in Sherman et al. (26). Yeast strains expressing wild type or temperature-sensitive Rieske iron-sulfur proteins were initially grown in 500 ml of minimal media (SD(26) supplemented with adenine, histidine, and tryptophan) in order to select against plasmid loss. The 500-ml cultures were used to inoculate carboys containing 17.5 liters of YPAD. These cultures were stirred, forcibly aerated, and incubated at 22 "C for all strains except JPJl-r203S, which was incubated at 17 "C. Cultures were harvested at a cell density of 1 X 10' cells ml-I; this cell density yields approximately 19 g (wet weight) of cell paste/ liter of culture. Harvested cells were washed twice with water, and the resulting cell paste was stored frozen at -70 "C.

Immediately prior to harvesting cells, sterile samples were removed to check for plasmid loss. These subsamples were diluted to lo3 cells ml-I, and 0.15 ml were spread on duplicate YPAD agar plates. After 3 days, the number of colonies was noted and colonies were replica plated onto YPAG and minimal media (SD supplemented as above). After 3 days the number of Gly' and Ura+ colonies were counted. As expected, all ura- colonies were gly-, 10% of strains JPJ1-R1, JPJ1- r851, JPJl-r102L, JPJl-l57S, and JPJl-rl75S had lost plasmids and reverted back to JPJ1. This rate is consistent with a 1% mitotic loss rate typical for yeast centromeric plasmids (27). Strain JPJ1-203s had a significantly higher frequency of plasmid loss, and 17% of the cells reverted to JPJ1.

Large Scale Preparation of Mitochondrial Membranes-Greater than 50 g of yeast cell paste were thawed and suspended at 4 "C in 50 mM K,HPO,, 0.9% KC1,lO mM EDTA containing 1.0 mM phenylmethylsulfonyl fluoride, and 0.5 mM diisopropylfluorophosphate to form a cell suspension of 0.3 g of cells m1-I. Cells were disrupted by high pressure shearing, at pressure differentials exceeding 25,000 psi, by two passes through a Stansted Cell Disrupter (Stansted Fluid Power, Ltd., Stansted, England). The disrupted cell suspension was collected in a stainless steel beaker sitting in wet ice, and the temperature of the suspension was monitored and not allowed to exceed 15 "C. The suspension was stirred until the temperature of the solution returned to 5 "C before the second passage was initiated. Typi-

cally, 80-90% of the yeast cells were broken as judged by microscopic observation.

Broken cells were fractionated by differential centrifugation (28). The resulting submitochondrial particles were washed twice in buffer A (0.25 M sucrose, 10 mM potassium phosphate, pH 7.4,l mM EDTA) containing 1 mM phenylmethylsulfonyl fluoride. The washed pellet was resuspended with one pellet volume of buffer A, after which two pellet volumes of glycerol were added. Glycerol diluted membranes were stored at -20 "C.

Preparation of Mitochondria with Lyticase-Yeast cells (from 500 ml of culture) were washed one time and resuspended in 75 ml of SPEB (0.9 M sorbitol, 50 mM potassium phosphate, pH 7.4, 10 mM EDTA, 14 mM /3-mercaptoethanol) to which 12 mg of lyticase previously suspended in 0.5 ml of water was added. The suspension was placed in an Erlenmeyer flask and incubated with shaking at 30 "C. Spheroplast formation was assayed by the degree of optical clarity observed after 50 pl of cell suspension was added to 500 pl of 10% SDS. Typically, incubations of 3-5 h were required to achieve efficient spheroplasting. Spheroplasts were pelleted by centrifugation at 3000 rpm for 5 min in a Sorval SS-34 rotor. The pellet was gently washed once with 75 ml of 0.9 M sorbitol, 50 mM potassium phosphate, pH 7.4, 10 mM EDTA. The washed spheroplasts were resuspended and homogenized with eight passes in a Teflon-glass homogenizer in 75 ml of 0.9% sodium chloride, 10 mM potassium phosphate, pH 7.4, 1 mM EDTA. The homogenized solution was centrifuged as before, and the supernatant was collected. The supernatant was centrifuged at 10,000 rpm for 20 min, and the pellet was washed twice with the buffered saline solution and finally resuspended in 2 ml of buffered saline. Membranes were stored at -70 "C. This method yields 10-20 mg of membrane protein from a 500-ml culture of cells.

Small Scale Preparation of Mitochondria-Yeast cells from a 5-ml culture were pelleted, washed twice with distilled water, and trans- ferred to a 1.5-ml microcentrifuge tube. Cells were then additionally washed with 1 ml of SPEB (see lyticase lysis method) and resuspended in 100 p1 of SPEB. Forty pl of 2 mg ml" zymolyase-lOOT in 1 M sorbitol was added, and after vortexing the tube was incubated for 30 min at 37 "C. The cell suspension was briefly centrifuged and the pellet was resuspended and washed once with ice-cold high-SEMB (1.3 M sorbitol, 0.5 M EDTA, 10 mM MES, pH 6.4,0.2% bovine serum albumin). The resulting cell pellet was resuspended in 0.5 ml of low- SEMB (0.3 M sorbitol, 0.5 M EDTA, 10 mM MES, pH 6.4, 0.2% bovine serum albumin). This suspension was incubated on ice for 30 min. High-SEMB (0.5 ml) was then added and after vortexing the tube was again incubated 30 min at 4 "C. This suspension was centrifuged at 1,000 X g for 5 min. The supernatant was decanted and recentrifuged at 16,000 X g for 5 min. The pellet containing approximately 700 pg of membrane protein was resuspended with 50 ml of buffer A/glycerol (1:l) and membranes stored at -20 "C.

This method was scaled up in order to isolate mitochondrial membranes from yeast grown to confluence on 100-mm diameter agar plates. Yeast were harvested by flooding the plates with water. The cell paste was suspended by scraping with a bent glass rod. The suspension was centrifuged, and pelleted cells were washed three times with ice-cold water. The cells were additionally washed with 5 ml of SPEB and resuspended in 1 ml of SPEB. Two-hundred pl of 2 mg ml" zymolyase solution was added, and the suspension was incubated as previously described. After incubation the cells were pelleted and washed with 3.5 ml of ice-cold high-SEMB. Washed cells were then resuspended vigorously in 5 ml of low-SEMB and incubated as described. Five ml of high-SEMB was added, and the suspension was incubated again and centrifuged as described above. The resulting membranes (containing 2-4 mg of protein) were resuspended in 1.0 ml of buffered glycerol as above.

Enzyme Assays-Ubiquinol-cytochrome c oxidoreductase activity was assayed (23) using the ubiquinol analog, 2,3-dimethoxy-5-methyl- 6-n-nonyl-1,4-henzoquinol. Unless specified otherwise in the figure legends, the assays were performed at pH 7.5 and 15 'C. The nonen- zymatic rate of cytochrome c reduction was determined by adding 33 PM 2,3-dimethoxy-5-methyl-6-n-nonyl-1,4-benz0quin~l, allowing the reaction to proceed for 5 s, after which the enzymatic reaction was initiated. Cytochrome c oxidase activity was assayed as described in Berry and Trumpower (29).

Rates of reduction or oxidation of cytochrome c were monitored in a dual wavelength spectrophotometer using the wavelength pair 550- 539 nm with the slit adjusted to a 1-mm band pass; zero-order reaction rates were calculated using an extinction coefficient of 21.5 mM" cm" for reduced minus oxidized cytochrome c. One-half of the concentrations of cytochrome b and cytochrome aa3 in the membranes

Temperature-sensitive Iron-Sulfur Protein Mutants 3725

was used to estimate the quantities of cytochrome bcl and cytochrome c oxidase complexes, respectively. Turnover numbers (Tn s-'), mol of cytochrome c reduced or oxidized/mol of respiratory complex/s, were calculated based on these estimates. Enzymatic activities listed in tables or depicted in figures are the average of duplicate assays. In all cases duplicate enzymatic rates varied by less than 10%.

Spectroscopy-Optical spectra were obtained on an Aminco DW- 2A spectrophotometer with a I-nm band pass. Data was collected on a digital storage oscilloscope (Nicolet Instrument Co.) interfaced through an optical encoder attached to the monochrometer drive such that data points were collected at 0.1-nm intervals.

Absorption difference spectra were obtained in the dual wavelength mode with the reference beam set at 539 nm. Cytochrome b and cytochrome aa3 concentrations were determined by dithionite-reduced minus ferricyanide-oxidized difference spectra using the extinction coefficients 6562-577 = 25.6 mM" cm" (30) and c605-630 = 13.1 mM" cm" (31), respectively. Aliquots of 0.5 M sodium dithionite in 50 mM borate buffer, pH 9.5, were used to reduce ferricyanide- oxidized samples.

Solutions of antimycin, myxothiazol, and UHDBT were prepared in dimethyl sulfoxide, and their concentrations were determined spectrophotometrically, using extinction coefficients emM = 4.8 at 320 nm (32), 10.5 at 313 nm (33), and 12.2 at 287 nm (34), respectively.

EPR spectra were obtained in the laboratory of Dr. Tomoko Ohnishi, Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia. Spectra were recorded using a Varian E 109 spectrophotometer coupled to a dedicated IBM-PC microcom- puter used for signal averaging. Spectra were obtained at a microwave frequency of 9.32 GHz, modulation frequency of 100 kHz, modulation amplitude of 1.25 millitesla, microwave power of 2 milliwatts, time constant of 0.128 s, scan rate of 25 millitesla min-', and a sample temperature of 15 K.

Protein Manipulations-Protein was measured by a modified Lowry method (35, 36). Polyacrylamide gel electrophoresis in the presence of SDS was carried out according to Laemmli (37), except that SDS was omitted from the gel and lower electrode buffer.

Proteins separated by polyacrylamide electrophoresis were trans-

ferred to nitrocellulose filters as described by Towbin et al. (38). Filters were probed with the monoclonal antibody bcl-A15, which recognizes both native and SDS-denatured forms of the iron-sulfur protein, according to Johnson et al. (39) as modified by Beckmann et al. (8).

RESULTS

Plasmids containing the five reconstructed ts-ripl- genes are diagramed in Fig. 1 of the preceding paper. These ripl- genes were reconstructed with mutant PstI-EcoRI fragments flanked by wild type 5' (HindIII-PstI) and 3' (EcoRI-SacI) fragments. As indicated in the expanded map, all genetic elements required for expression of the ripl- genes are pre- served (8). Plasmid pRG415 contains yeast centromeric and autonomously replicating sequences and is thus stably main- tained at one copy/transformed cell. These plasmids were used to transform strain JPJl (see Table I of the previous paper for strain designations).

Mitochondrial membranes isolated from yeast strains grown at permissive temperatures, 22 "C for all strains except JPJl-r203S, which was grown at 17 "C, expressing wild type or temperature-sensitive Rieske iron-sulfur proteins have been characterized by a variety of biochemical methods. Mem- branes were examined for their cytochrome and iron-sulfur protein content. Ubiquinol-cytochrome c oxidoreductase activities in the isolated membranes were assayed under a variety of conditions in uitro. Finally, the effects of temperature-sensitive mutations on post-translational processing of the iron-sulfur protein in vivo were examined by immunoblot analyses of mitochondrial membranes from cells grown at various temperatures.

FIG. 1. Absorption difference spectra (dithionite-reduced minus ferricyanide-oxidized) of mitochondria membranes from wild type yeast W303- 1 A, iron-sulfur protein deletion strain JPJ1, and JPJl strains expressing either wild type or mutant iron-sulfur proteins. Mi- tochondrial membranes from yeast cells grown to confluence on 100-mm Petri plates containing solidified YPAD media at permissive temperatures, 22 "C for all strains except JPJl-r203S which was grown at 17 "C, were isolated by the scaled up mini-prep protocol. Mem- branes were diluted to 2.0 mg ml" with a solution of 1:l buffer A and glycerol. Triton X-100 was added to a final con- centration of 0.1% to disperse the membranes and significantly reduce spectral noise.

h (nm) I

550 570 590 610

550 3 JPJl A

551 4

JPJl-r851

551 2 h 550 1

567 I

JPJ1-R1

551 3

f l st.1 a JPJ1-rl57S


Optical Spectroscopy and Cytochrome c Reductase Actiuity- Optical spectra of membranes from cells grown on solidified YPAD media are shown in Fig. 1. Typical cytochrome absorbance spectra with C-type ( Xmax 550-553 nm), b-type (X,,, 561- 566 nm), and a-type (X,,, 603-605 nm) hemes are observed for all strains except JPJ1. Strain JPJl has approximately one-third of the wild type cytochrome b content and also has less cytochrome cl, which is seen by the shift in the absorbance maximum of C-type hemes (551.h550.3). The mutant forms of the iron-sulfur protein do not significantly affect the concentrations of membrane cytochromes, when the yeast are grown at permissive temperatures on solid media, as summa- rized in Table I. However, in liquid culture the concentrations of cytochromes are lower than on solid media, and there was a further diminution of cytochromes in all of the mutants relative to JPJ1-R1.

Ubiquinol-cytochrome c oxidoreductase activities of membranes from cells grown at permissive temperatures on solid YPAD media are shown in Table I. Membranes from wild type strain W303-1A and strain JPJ1-R1, the deletion strain

TABLE I Spectral characterization and enzymatic activities of mitochondrial membranes isolated from cells at permissive temperatures, 22 "C for

all strains except JPJI-r203S which was grown at 17 "C, on solidified or in liquid YPAD media

cytochrome c oxidoreductase; Tn, turnover numbers. The abbreviations used are: Cyt., cytochrome; QCR, ubiquinol-

Strain [bel complex] [Cyt. oxidase] QCR Cyt. oxidase

Solid culture W303-1A JPJl

JPJl-r851 JPJ1-rl02L JPJ1-r157S JPJ1-r175S JPJl-r203S

Liquid culturea

JPJI-r85I JPJ1-rl02L JPJ1-r157S JPJ1-rl75S JPJl-r203S

JPJ1-R1

JPJ1-R1

pmol mg"

287 135 306 353 282 275 308 291

98.4 53.3 52.5 45.1 66.5 38.9

pmol mg" Tn s-'

124 216.9 148 0.0 155 188.5 190 125.7 144 146.4 132 80.6 175 50.1 164 63.6

38.4 46.0 12.1 30.6 21.6 28.9 15.3 8.3 36.2 10.9 9.6 3.7

Tn s-l

95.4 240.8 155.8 146.0 127.7 110.9 196.2 121.4

128.5 263.7 104.5 130.8 174.6 60.2

Cells harvested at a density of -1 X 10' cells m1-l.

FIG. 2. Electron paramagnetic resonance spectra of mitochondrial membranes from JPJl strains expressing wild type or mutant iron- sulfur proteins. Mitochondrial membranes from yeast grown in 18-liter cultures were pelleted by centrifugation for 60 min at 100,000 x g. EPR tubes were filled with 0.3 ml of pelleted membrane preparations, and each preparation was reduced with 6 pl of 0.5 M sodium dithionite in 50 mM Tris-propionate buffer, pH 9.5, and immediately frozen. The traces are averages of four scans.

carrying plasmid encoded wild type iron-sulfur protein gene, have similar ubiquinol-cytochrome c oxidoreductase turnover numbers. The strains expressing mutant iron-sulfur proteins have varying cytochrome c reductase turnover numbers, rang- ing from 77 to 26% of the activity in membranes from JPJ1- R1 (Table I). Although there were quantitative differences in their sensitivity to respiratory inhibitors, all of the membranes were inhibited by antimycin, myxothiazol, and UHDBT.

To further characterize the effects of mutant iron-sulfur proteins, membranes were obtained from cells grown in 18 liters of liquid YPAD media at permissive temperatures (22 "C for all strains except JPJl-r203S, which was grown at 17 "C) and harvested during late log phase. The cytochrome contents and enzymatic activities of these membranes are listed in Table I.

EPR Spectroscopy-Mitochondrial membranes from the large scale liquid cultures were characterized by electron paramagnetic resonance spectroscopy and the spectra are shown in Fig. 2. These spectra were difficult to obtain due to the low iron-sulfur protein concentrations in yeast membranes (10% of that normally observed in mammalian mitochondrial membranes). The resonance at g = 1.89 is characteristic of the Rieske iron-sulfur protein. As can be seen in the traces, all membranes contain a discernible amount of iron-sulfur protein, which is crudely quantitated in Table 11. The amounts of iron-sulfur protein within these membranes correspond roughly with the amounts of cytochrome b. No major changes of the EPR signals or line shapes are evident, although higher resolution spectra would be required to cor- roborate this preliminary observation.

Effects of Temperature and pH-Incubation of membranes at higher, nonpermissive temperatures led to an exponential loss of enzymatic activity (data not shown). Thermal stability profiles of ubiquinol-cytochrome c oxidoreductase in membranes from each ts-ripl- strain are shown in Fig. 3. Mem- branes from strains expressing mutant iron-sulfur proteins exhibited significantly less thermostability than wild type membranes. Membranes from JPJl-r203 exhibited the great- est temperature sensitivity.

The effect of pH on ubiquinol-cytochrome c oxidoreductase activities was examined, and the results are shown in Fig. 4. The control strain, JPJ1-R1, shows a slight pH dependence, with the rate of cytochrome c reductase activity declining approximately 30% from pH 8 to 6.5. Membranes from three

JPJ I -r 157s

I q - ' 8 9 I I/ I

Temperature-sensitive Iron-Sulfur Protein Mutants

TABLE I1 Electron paramagnetic resonance spectroscopy of mitochondrial

membrane preparations The abbreviations used are: Cyt., cytochrome; ISP, iron-sulfur

Strain Protein [Cyt. b] [ISP]" [Cyt. b] mg" [ISP] mg" protein.

mg ml" p m d mg" units mg" 96 %

JPJ1-R1 48.7 196.8 112.9 100 100 JPJl-r851 64.5 106.6 95.6 54 85 JPJ1-rl02L 77.0 105.0 48.6 53 43 JPJl-rl57S 68.3 90.2 39.0 46 35 JPJ1-rl75S 63.6 133.0 59.0 68 52 JPJl-r203S 57.7 77.8 37.6 40 33 ' Arbitrary units = depth of trough in the g, 1.90 resonance relative

to high field base line divided by mg protein X 100.

60

3 40 -

20

T h r 8 5 * I l e , 0

15 25 35 4S 55 65

'. Preincubation Temveroiure ('C)

Pro 102 -+ Leu

15 25 35 45 55 65 Preincabatio. Temperature (OC)

" , . I

6 5 7 0 1 5 8.0 vn

2o 1 Gly 157 -+ Ser

6 5 7.0 7 5 8 0 PH

3121

P r o 1 0 2 -+ Leu 0

6.5 7 0 7 5 8 .o vn

G l y 1 7 5 + S e r

6.5 70 7 .S 8.0 PH

2: 6.5 L 7 0 P r o 2 0 3 7.5 + Ser

PH 8 0

FIG. 4. Effect of pH on ubiquinol-cytochrome c oxidoreductase in mitochondrial membranes from JPJl strains expressing either wild type or mutant iron-sulfur proteins. Ubiquinol- cytochrome c oxidoreductase activities of mitochondrial membranes, isolated from 18 cultures, were determined at the indicated pH values. In each plot mitochondrial membranes from strain JPJ1-R1 (open squares, solid l i n e ) and a JPJl strain expressing mutant iron-sulfur protein (solid diamonds, dashed line) are compared. The wild type amino acid and mutant induced change being compared are indicated in each plot. Activities are plotted as a percentage of the maximal activity observed for each strain. The quinol-cytochrome c reductase activities of JPJ1-R1 and the five mutants grown in liquid culture and assayed at pH 7.5 are shown in Table I.

2: 15 u P r o 2 0 3 25 -+ 35 Ser \, 45 ". 55 65

Prcincmbalien Temperature ('C)

FIG. 3. Thermal stability of ubiquinol-cytochrome c oxidoreductase in mitochondria membranes from JPJl strains expressing either wild type or mutant iron-sulfur proteins. Mi- tochondrial membranes, isolated from 18-liter cultures, were prein- cubated for 15 min at the temperatures indicated. Ubiquinol- cytochrome c oxidoreductase activity was assayed immediately there- after at 15 "C. In each plot mitochondrial membranes from strain JPJ1-R1 (open squares, solid l i n e ) and a JPJl strain expressing mutant iron-sulfur protein (solid diamonds, dashed l i n e ) are compared. The wild type amino acid and mutant induced change being compared are indicated in each plot. Activities are plotted as a percentage of the maximal activity observed for each strain. The quinol-cytochrome c reductase activities of JPJ1-R1 and the five

Table I. mutants grown in liquid culture and assayed at pH 7.5 are shown in

of five mutant strains, 102L, 157S, and 175S, exhibited nearly identical pH profiles as compared to those from strain JPJ1- R1. However, at pH values below 7, these three mutant membranes show decreased enzymatic activities compared to JPJ1-R1. Mutation 851 resulted in an iron-sulfur protein which is significantly more sensitive to lower pH than the wild type protein. The possible significance of this sensitivity to pH is discussed below.

Inhibitor Sensitivities-Although we selected for tempera-

ture-sensitive mutations, we expected that mitochondrial membranes from these mutants may show differing responses to inhibitors acting in the cytochrome bcl complex. We hy- pothesized that inhibitors which act proximal to or on the iron-sulfur protein would be sensitive to changes in the structure of the protein, whereas those which act distal to the iron- sulfur protein would not. Therefore, we examined the effects of antimycin, myxothiazol, and UHDBT on ubiquinol-cytochrome c oxidoreductase activities in mitochondrial membranes from strains expressing wild type and mutant iron- sulfur proteins.

Antimycin, which inhibits at center N, at a site distal to the iron-sulfur protein, inhibited all membranes identically, resulting in virtually superimposable inhibition curves (Fig. 5 ) . The amounts of antimycin required to achieve 50% inhibition and the extrapolated amounts required for 100% inhibition are listed in Table 111.

Ubiquinol-cytochrome c oxidoreductase activities in membranes of all five strains carrying mutant iron-sulfur proteins exhibit a slight but significant resistance to myxothiazol inhibition, as shown in Fig. 6 and Table 111. Interestingly, although the mutations are spread over a large portion of the iron-sulfur protein, they all confer nearly identical resistance to myxothiazol.

UHDBT inhibition data are presented in Fig. 7 and Table 111. The inhibitory effect of UHDBT is very sensitive to assay


Thr85 "t I l e

2 40

20

0 mol ANTllmol bcl camplax

I O 20 30

I!;;., 1 Pro I02 4 Leu

40

20

x. 0 0 I O 20 30

mal ANTllrnol bcl cemplex

100 Thr85 -b I le jq-J Thr85 -b I le

60

2 40

20 '. 0 0 4 8 I 2 16

mol MVXOlmd bcl complcx

'1 30 0 I O 20 30

P I 0 I O 20 30

mol LNTllmoI bc1 complex

FIG. 5. Effect of antimycin on ubiquinol-cytochrome c oxidoreductase in mitochondrial membranes from JPJl strains expressing either wild type or mutant iron-sulfur proteins. Ubiquinol-cytochrome c oxidoreductase activities of mitochondrial membranes isolated from 18-liter cultures were determined at increasing antimycin to bcl complex ratios. In each plot mitochondrial membranes from strain JPJ1-R1 (open squares, solid line) and a JPJl strain expressing mutant iron-sulfur protein (solid diamonds, dashed line) are compared. The wild type amino acid and mutant induced change being compared are indicated in each plot. Experimentally determined inhibitor to complex ratios with 100 to 40% of control activity, the linear portions of the inhibition curves, are plotted to allow extrapolation to 100% inhibition. The quinol-cytochrome c reductase activities of JPJ1-Rl and the five mutants grown in liquid culture and assayed at pH 7.5 are shown in Table I.

TABLE I11 Inhibitor concentrations required to achieve 50 and 100% inhibition of ubiquinol-cytochrome c oxidoreductase activity in mitochondrial

membranes from JPJl-R1 and temperature-sensitive mutant strains UHDBT Myxothiazol Antimycin

Strain 50% 100% 50% 100% 50% 100%

inhibitorlmole enzyme

JPJ1-R1 27.3 861 4.18 8.55 11.35 23.84 JPJ1-~851 5.7 42 7.24 14.58 13.46 26.48 JPJ1-rl02L 31.0 770 6.02 12.25 11.17 22.20 JPJ1-r157S 56.7 2148 6.26 12.58 11.97 22.29 JPJl-rI75S 50.8 1569 6.37 13.24 10.25 19.81 JPJl-r203S 25.3 535 5.67 12.14 12.96 24.09

pH. In intact mitochondrial membranes at pH 7.5, UHDBT is normally not as potent an inhibitor as either antimycin or myxothiazol (see Table I11 strain JPJ1-R1) (25). Mutation 851 induces a substantial increase in sensitivity to UHDBT; only 20% as much UHDBT is required to achieve 50% inhibition of ubiquinol-cytochrome c oxidoreductase activity in membranes from JPJl-r851 compared to membranes from JPJl-Rl.* As a result of this mutation, UHDBT inhibits 50%

Pro IO2 -b Leu

80

I : : r

Pro IO2 -b Leu

60 e'*,.

40

20

'. 0 0 4 8 12

mol MVXOlm~l bcl complex

'The effect of pH on activity and UHDBT hypersensitivity of JPJl-r851 were also observed when succinate-cytochrome c activity of mitochondrial membranes was measured.

. - 0 4 6 12

mol MVXOlm~l bcl complex

IO0

BO

,z 60 c + p 40

20

0

'~ 0 . , . , . : . 0 4 8 12 16

mol MYXOlnol b c I complex

FIG. 6. Effect of myxothiazol on ubiquinol-cytochrome c oxidoreductase in mitochondrial membranes from JPJl strains expressing either wild type or mutant iron-sulfur proteins. Ubiquinol-cytochrome c oxidoreductase activities of mitochondrial membranes isolated from 18-liter cultures were determined at increasing myxothiazol to bcl complex ratios. In each plot mitochondrial membranes from strain JPJ1-R1 (open squares, solid line) and a J P J l strain expressing mutant iron-sulfur protein (solid diamonds, dashed line) are compared. The wild type amino acid and mutant-induced change being compared are indicated in each plot. Experimentally determined inhibitor to complex ratios with 100 to 40% of control activity, the linear portions of the inhibition curves, are plotted to allow extrapolation to 100% inhibition. The quinol- cytochrome c reductase activities of JPJ1-R1 and the five mutants grown in liquid culture and assayed at pH 7.5 are shown in Table I.

of the cytochrome c reductase activity of these membranes at lower concentrations than either antimycin or myxothiazol (Table 111). Two mutations, 157s and 175S, appear to confer a slight resistance to UHDBT.

Effects on Post-translational Processing-Since temperature-sensitive mutations are expected to affect the folding of newly synthesized protein, we also examined the effects of these mutations on the processing of the iron-sulfur protein. Mitochondrial membranes from cells expressing temperature- sensitive iron-sulfur proteins were solubilized and electropho- retically separated as described in Fig. 8. These membranes were from cells grown at 30 "C in liquid YPAD media. This is a growth temperature at which all strains except JPJ1- 1-203s would be expected to form functional iron-sulfur proteins. As can be seen, a protein band of intermediate size is observed between the precursor and mature iron-sulfur protein in all of the lanes except ripl-203s.

Mutations 102L and 175S, and to a lesser extent 851 and 157S, all affected post-translational processing of iron-sulfur protein (Fig. 8). This is demonstrated by relative increases in the pre-iron-sulfur protein bands. Processing of the protein containing the 102L mutation is especially inhibited. Al- though similar amounts of total membrane protein were ap- plied to each lane, membranes isolated from strain JPJ1-

Temperature-sensitive Iron-Sulfur Protein Mutants 3729 ~~~1 80 60 '. . * P r o 1 02 + L e u

40

20 s. .o

0 0 2 4 6 8

1. mol U H D B T l m l 8 c 1 Comvlex

- . 0 i i 6 d -0 i 6 e

I n mol UHDBTlnml bc, complex 1. ~1 UHDBTlnd bc1 cemplex

04 . , 0 2 4 6 e

11 m 1 UHDBTlnol be, complex

FIG. 7. Effect of UHDBT on ubiquinol-cytochrome c oxidoreductase in mitochondrial membranes from JPJl strains expressing either wild type or mutant iron-sulfur proteins. Ubiquinol-cytochrome c oxidoreductase activities of mitochondrial membranes, isolated from 18-liter cultures were determined at pH 7.5 at increasing UHDBT to bcl complex ratios. In each plot mitochondrial membranes from strain JPJ1-Rl (open squares, solid line) and a JPJl strain expressing mutant iron-sulfur protein (solid diamonds, dashed line) are compared. The wild type amino acid and mutant-induced change being compared are indicated in each plot. Experimentally determined inhibitor to complex ratios with 100 to 40% of control activity, the linear portions of the inhibition curves, are plotted to allow extrapolation to 100% inhibition. The quinol- cytochrome c reductase activities of JPJ1-R1 and the five mutants grown in liquid culture and assayed at pH 7.5 are shown in Table I.

r203S exhibit immunoreactive bands which are present in barely detectable amounts. This is consistent with the growth characteristics of this strain at the various temperatures, as described in the preceding paper.

The data shown in Fig. 9 compare the state of iron-sulfur protein processing in mitochondrial membranes isolated from large scale membrane preparations, which were used for enzymatic and EPR analyses, and membranes isolated from yeast cells grown at intermediate and nonpermissive temperatures. Washed mitochondrial membranes were examined for the relative concentrations of precursor and mature iron- sulfur protein. Due to differing levels of total iron-sulfur protein and differing growth conditions, it is necessary to normalize the relative intensities of precursor to the intensity of mature iron-sulfur protein in each lane. I t is also most informative to compare membranes isolated from cells grown under similar conditions. The data clearly show that membranes isolated from cells grown a t permissive temperatures, with the exception of mutant 102L membranes, contain mainly processed, mature iron-sulfur protein (Fig. 9). Muta- tion 102L diminishes the ability of the iron-sulfur protein to be processed efficiently. Even at 22 "C, this mutation affects the relative amount of precursor band as compared to wild type membranes (RIP1).

FIG. 8. Immunoblot analysis of mitochondrial membranes from JPJl strains expressing either wild type or mutant iron- sulfur proteins. Mitochondrial membranes from cells grown at 30 "C in YPAD media are compared. Membranes were isolated from cells by the lyticase method. Approximately 40 pg of membrane proteins in 10 pl of 1 X upper gel buffer were denatured and solubilized in the presence of 5% 8-mercaptoethanol and 1% SDS. After heating 2 min at 95 "C 4 grains of sucrose and 1 pl of 0.2% bromphenol blue were added. These denatured samples were electrophoresed through 12.5% polyacrylamide gels and electroblotted to nitrocellulose membranes. Blots were probed with monoclonal antibody bcl-A15 (8). The positions of precursor ( p - I S P ) and mature (m-ISP) forms of iron-sulfur protein are indicated on the left.

Each of the other mutations reduces the relative concentrations of the mature iron-sulfur proteins at higher temperatures, while concomitant increases in the levels of preproteins are observed. For example, with mutation 851, the relative amounts of pre and mature proteins a t 30 "C are similar to those observed in the wild type membranes (RIP1). However, a t 37 "C there is an obvious decrease in processed mature iron-sulfur protein compared to preprotein (Fig. 9). This same pattern is observed for mutations 157s, 175S, and 203s.

DISCUSSION

The decreases in cytochromes b and c, in strain JPJl (Fig. 1) imply that iron-sulfur protein is required for the mainte- nance of wild type levels of these cytochromes. I t is unlikely that the iron-sulfur protein, a nonregulatory protein, directly affects the rates of synthesis of cytochromes b and cl. The most plausible explanation for the decreased levels of cytochromes in JPJl is that the partially assembled b c ~ complex, that is lacking iron-sulfur protein, is subject to increased rates of degradation.

The ubiquinol-cytochrome c oxidoreductase activities in the isolated membranes (Table I) are consistent with the observed growth rates of the strains from which they originate (see Table IV of the preceding paper); low enzymatic activity manifests an increase in the doubling time. Not surprisingly, mutations closest to the 2Fe-2S cluster-binding sites (157S, 175S, and 203S, see Fig. 9 in the preceding paper) have the lowest ubiquinol-cytochrome c oxidoreductase activities (Ta- ble I). These data are consistent with the high degree of amino acid sequence conservation exhibited within these regions (see Fig. 8 in the preceding paper).

Culture conditions also affect cytochrome content and enzymatic activity of the membranes (Table I). This is most probably due to different levels of glucose repression, although the exact reason is not known. Regardless of the culture conditions, membranes from strains expressing mutant iron- sulfur proteins had lower ubiquinol-cytochrome c oxidoreductase activities, expressed as turnover numbers, and the percentage differences compared to JPJ1-R1 were nearly the same (Table I).

Each of the five ts-ripl- mutations exhibited pleiotropic


FIG. 9. Effect of growth temperature on iron-sulfur protein processing in mitochondrial membranes from JPJl strains expressing either wild type or mutant iron-sulfur proteins. Mitochondrial membranes were isolated from cells grown in YPAD media at permissive temperatures (22 or 17 "C), 30 and 37 'C. Membranes from cells grown at permissive temperatures were from 18-liter cultures. Mitochon- drial membranes from 5-ml yeast cultures grown at 30 and 37 "C were isolated by the small scale protocol. Membrane samples were denatured and treated as described in Fig. 8. The relative positions of precursor ( p - I S P ) and mature (m- ISP) forms of iron-sulfur protein are indicated on the left. The first lanes (bel) in both blots contain 1 pg of purified yeast cytochrome bc, complex.

p-ISP - m-ISP -

p-ISP + m-ISP +

RIP1 rlpl-851 rlpl-lO2L -7

bc1 22' 30' 37' ' 22' 30' 37' ' I 22' 30' 37' '

effects. Although the mutations had several in vitro phenotypes in common, each exhibited unique properties. The ubiquinol-cytochrome c oxidoreductase activities of mitochondrial membranes from all strains expressing mutant ts-ripl- genes exhibited decreased thermal stability as compared with wild type membranes (Fig. 3). Since the iron-sulfur protein is a subunit of a large integral membrane lipoprotein complex, each of the mutations must compromise the stability of the iron-sulfur protein sufficiently to override stabilizing influ- ences of intracomplex protein-protein interactions.

Respiratory inhibitors proved to be effective probes of the environment surrounding the iron-sulfur protein. The results obtained with antimycin (Fig. 5) indicate that structural changes in the mutant iron-sulfur proteins do not induce changes in the antimycin-binding site at center N, which is thought to be on cytochrome b, distal to the site at which cytochrome b interacts with the iron-sulfur protein (22).

Myxothiazol binds to cytochrome b, but displaces ubiquinone from a binding site that is on or near the iron-sulfur protein (24). This suggests that cytochrome b and the iron- sulfur protein must interact. All five temperature-sensitive mutations confer quantitatively similar resistance to myxothiazol, even though they are spread throughout the protein. We interpret this result as indicating that all of these mutations affect a small global change in the iron-sulfur protein structure, so that binding of myxothiazol to cytochrome b does not transmit a conformational change to the ubiquinol- binding site on the mutant iron-sulfur proteins as effectively as in the wild type protein.

UHDBT is a hydrophobic structural analog of ubiquinone which binds preferentially to reduced iron-sulfur protein and displaces ubiquinone from a binding site that is on or near the iron-sulfur protein (25). Thus, a mutation which decreases the hydrophobicity around the UHDBT-binding site or that preferentially stabilizes oxidized iron-sulfur protein would decrease the affinity for UHDBT. Either of these possibilities could explain the apparent in uitro resistance to UHDBT conferred by mutations 157s and 175s. Potentiometric titra- tions of the 2Fe-2S cluster in these mutant proteins are needed to further clarify this initial observation.

The increased sensitivity to UHDBT induced by mutation

851 could be due to increased hydrophobicity around the UHDBT-binding site. Edwards et al. (40) demonstrated that UHDBT homologs with longer side chains are more effective inhibitors. However, an increased hydrophobic environment within the protein should not discriminate between ubiquinone and UHDBT, both of which are extremely hydrophobic molecules. Therefore, it is not clear why an increase in hydrophobicity should favor UHDBT over ubiquinone. A local change of hydrophobicity also does not explain the altered pH dependence of activity in mutant 851 (Fig. 4).

A mutation that induces a structural change which preferentially stabilizes reduced iron-sulfur protein would increase the affinity for UHDBT. Likewise, a mutation which impedes electron transfer between the iron-sulfur protein and cytochrome cl, or which increases the rate of electron transfer from ubiquinol to the iron-sulfur protein would effectively increase the steady state levels of reduced iron-sulfur protein. In all of these cases UHDBT would be expected to bind with greater affinity. These possibilities provide plausible expla- nations for the observed UHDBT hypersensitivity exhibited by mutation 851, but they do not readily explain the additional observations associated with this mutation.

An alternative explanation for the UHDBT hypersensitivity observed with mutation 851 is that threonine 85 is located at or near the ubiquinol-binding site on the iron-sulfur protein and that mutation 851 disrupts this site. This model is supported by four experimental findings. Mutation 851 confers extreme pH sensitivity (Fig. 4), hypersensitivity to UHDBT inhibition (Fig. 7), lower enzymatic activity (Table I), and is located at the iron-sulfur protein/membrane interface.

This model also suggests that threonine 85 may be involved in catalysis within the ubiquinol-binding site, Rich (41) has suggested that the rate of oxidation of ubiquinol is determined by a residue capable of hydrogen bonding with one of the quinol hydroxyl groups. I t is possible that threonine 85 in the wild type protein catalyzes oxidation of ubiquinol by forming a hydrogen bond, thereby promoting the formation of ubiquinol anion, an otherwise slow reaction. In the absence of threonine 85, oxidation of ubiquinol would be retarded, and this reaction would become more sensitive to the pH of the medium. Mutant 851 exhibits a marked decrease in enzymatic

Temperature-sensitive Iron-Sulfur Protein Mutants 3731

activity at lower pH compared to wild type (Fig. 4). Many imported mitochondrial proteins contain NHp-ter-

minal amino acid sequences that are proteolytically cleaved during a maturation process that often involves the concomitant insertion of prosthetic groups (13-15). The mitochondrial import and subsequent processing reactions of the Neu- rospora crassa Rieske iron-sulfur protein have been well characterized (16), and a two-step processing pathway has been demonstrated. Whether a similar pathway, involving a two- step proteolytic maturation, is operative in yeast is not yet established.

Our results indicate that several temperature-sensitive mutations, especially 102L, mimic the treatment of isolated mitochondria with EDTA and orthophenanthroline. The agents block processing in vitro (16) but do not block translocation of proteins into the matrix of the mitochondria (42). Since it is likely that the iron-sulfur cluster plays an important role in the conformation of the Rieske protein, our results further suggest that the 2Fe-2S cluster may be inserted prior to proteolytic processing. Mutations which retard the insertion of the 2Fe-2S cluster would also retard proteolytic maturation.

These alterations in post-translational processing could also be explained by three less likely alternatives. First, mutant preproteins could be unrecognizable by a mitochondrial surface receptor. This seems unlikely since we examined washed mitochondria (Figs. 8 and 9), and proteins not tightly associated would not be observed. Also, mutations spread throughout the protein lead to the same lack of processing. It is hard to imagine that single point mutations far removed from the presequence could affect the receptor interaction, since there are numerous examples of fusion proteins containing mitochondrial leader sequences attached to nonmitochon- drial proteins which are imported. It seems that, in most instances, the presequence alone provides sufficient infor- mation for recognition and import (15).

Proteins imported into the mitochondria undergo unfolding, and proteins that are conformationally “locked” are completely translocated (43). Either or both the unfolding and subsequent translocation are ATP dependent (44). Thus, our results could be explained if mutations somehow blocked the translocation process. The fact that there is no indication of protease damage, except in mutant 203S, indicates that the iron-sulfur proteins are most probably translocated into a protease inaccessible space. The protease digestion of ripl- 203s suggests that this protein is very unstable, and degraded either en route to the mitochondria, or by mitochondrial proteases.

The last alternative to explain the accumulation of pre- iron-sulfur protein is that these mutations alter the protease recognition site and thereby block processing. This also seems unlikely, since mutations spread throughout the protein lead to a similar lack of processing.

The retarded rates of processing observed at nonpermissive temperatures suggest that folding and insertion of the 2Fe-2S cluster into the imported iron-sulfur protein are obligatory prior to proteolytic processing. The intermediate band seen in Fig. 8 provides support for a two-step processing pathway in yeast (12), but radiolabeled pulse-chase experiments are required to confirm these results.

The carboxyl terminus of the iron-sulfur protein was found

to be important for stabilization of the protein. Mutation 203S, 12 residues from the carboxyl terminus, produced pro- found temperature sensitivity (Fig. 3), resulting in rapid degradation of the protein at temperatures above 17 “C. This region of the protein may be essential in blocking degradation of pre-iron-sulfur protein by cytoplasmic proteases, as the protein is imported into the mitochondria, or may be a “degradation signal” in the mature protein which tags the iron- sulfur protein for turnover. The significant decrease in thermostability observed with this protein, even after maturation, suggests a critical role for the carboxyl terminus in mainte- nance of protein structure, although the isolation of additional mutations within this region will be necessary to confirm this observation.

Acknowledgment-We thank Dr. Tomoko Ohnishi, Department of Biochemistry and Biophysics, University of Pennsylvania, Philadel- phia, for the use of her EPR spectrometer.

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journal of vol. 264, no. 7, of 5, pp. 1989 by for …vol. 264, no. 7, issue of march 5, pp....

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