ARCHIVES

OF

BIOCHEMISTRY

Mitochondrial

AND

BIOPHYSICS

175, 606-617 (1976)

Biogenesis during Fungal Spore Germination: of Mitochondrial Protein Synthesis in Vivol R. BRAMBL

Department

of Plant Pathokgy,

AND

The University Received

Products

B. HANDSCHIN of Minnesota,

February

Saint Paul, Minnesota

55108

20, 1976

Germinated spores of the fungus Botryodiplodia theobromae synthesized about eight to ten mitochondrial proteins in the presence of cycloheximide as shown by acrylamidegel electrophoresis of 13Hlleucine-labeled, detergent-solubilized mitochondrial extracts. The apparent molecular aeights of these proteins (whose synthesis was blocked by chloramphenicol) ranged from 85,100 to 8400. The 8400 molecular weight protein was soluble in chloroform-methanol, while other mitochondrial proteins (including also those proteins synthesized only in the absence of cycloheximide) were insoluble in this solvent system. Treatment of intact mitochondria with trichloroacetic acid before detergent extraction caused conversion of the higher molecular weight radioactivity to the 8400 molecular weight form. Labeling kinetics of 300-min (germinated) spores indicated that the 8400 molecular weight protein was the first to contain 13Hlleucine, and with increasing time of incubation the label appeared in the higher molecular weight proteins. The mitochondrial ribosomes of the spores were not active during the first 60 min of germination, although products of the cytoplasmic ribosomes were incorporated into or associated with mitochondria during this interval; during the second 60-min interval of germination, function of mitochondrial ribosomes was observed by synthesis of the 8400 molecular weight, chloroform-methanol-soluble protein. In three subsequent 60min intervals of germination, radioactivity from [3H]leucine was incorporated into the higher molecular weight proteins. The significance of this mitochondrial protein synthesis in relation to other metabolic activities of the germinating spores is discussed.

The germination of fungal spores represents a useful experimental system for study of certain biochemical processes of eukaryotic cell development and organelle biogenesis. In our studies of spore germination we are especially interested in analysis of the cooperation and coordination that must exist between the nuclear and mitochondrial genetic systems for the functional organization of mitochondria. We wish to identify eventually the contributions of each genetic system toward assembly and function of the mitochondrial respiratory apparatus as the spores resume rapid metabolic activity during germination and to establish the temporal sequence of synthesis and assembly of the

cytoplasmic and mitochondrial ribosome products into newly organized mitochondria. In previous reports, we presented evidence that the mitochondria of ungerminated conidiospores of Botryodiplodia theobromae contain a preserved, potentially functional aerobic respiratory system which requires cytoplasmic ribosome function for initiation of respiratory activity (1). Translation of a preserved messenger RNA on cytoplasmic ribosomes is required for both respiration and germination of these spores (1, 2). Once activated, the latent cyanide-sensitive respiratory system appears to support the respiratory demand of the spores until new mitochondria presumably are first organized at about 150 min of germination. Thus, these spores can begin germination in the ab-

’ Paper NO. 9418, Scientific Journal Series, Minnesota Agricultural Experiment Station, Saint Paul, Minn. 606 Copyright 0 1976 by Academic press, Inc. All rights of reproduction in any form reserved.

SPORE

MITOCHONDRIAL

sence of mitochondrial genetic system activity, although function of the mitochondrial genetic system is required for cell growth beyond the first stages of germination (1, 3). Several studies have described properties of some of the products of the mitochondrial ribosomes after in. uiuo synthesis of the proteins in the presence of cycloheximide, an inhibitor of the cytoplasmic ribosomes. In physiologically mature cells of Saccharomyces (4, 5) and Neurospora (681, evidence indicates that proteins synthesized on mitochondrial ribosomes can be fractionated by their solubility in chloroform-methanol; a portion of the mitochondrial ribosome products are soluble in this solvent, suggesting a distinctive hydrophobicity of at least some of the mitochondrial proteins. In this present report is described a series of experiments undertaken to examine the nature of the mitochondrial proteins synthesized during fungal spore germination, to describe some of their properties, and to measure the timing of their synthesis as the cells undergo a developmental transition from metabolic quiescence to active growth with highly active mitochondrial aerobic respiration. MATERIALS

AND

METHODS

Techniques for production and germination of the spores and for preparation of the mitochondrial fraction have been described in detail previously (1). The spores were labeled with [DH]leucine (specific activities of 81.3, 47.6, or 25 Cilmmol, from New ICN) or England Nuclear Corp. or [3Hlphenylalanine (53 Ci/mmol, New England Nuclear Corp.) at concentrations of 0.2 $X/ml of spore suspension and with [14Clleucine (240 mCi/mmol, New England Nuclear Corp.) at 0.02 &i/ml of suspension. The concentrations of cycloheximide and chloramphenicol (Sigma) were 100 pg/ml and 3 mg/ ml, respectively; cycloheximide was added to spore suspensions 5 min before addition of the labeled amino acid. The mitochondrial pellets were resuspended and extracted with cold 90% methanol for 30 min before fractionation. For preparation of the SDS-soluble the methanol-extracted mitochondria fraction,* were suspended in a K-phosphate (10 mM) buffer solution (pH 6.5) containing 1% SDS, 1% 2-mercap’ Abbreviation fate.

used: SDS, sodium

dodecyl

sul-

PROTEIN

SYNTHESIS

607

toethanol, and 10% glycerol. These mitochondrial protein suspensions were incubated at 60°C for at least 12 h and then boiled for 2 min and centrifuged to remove insoluble material. Pyronin Y was added to these samples as a tracking dye in the subsequent electrophoresis. For the chloroform-methanol extraction of the methanol-extracted mitochondria, the mitochondria were suspended in a 2:l (v/v) mixture of these solvents, and the mixture was heated at 50°C for 30 min. The suspension was centrifuged and the resulting pellet was reextracted; the supernatant fluids were pooled, dried under nitrogen gas at 5o”C, and the chloroform-methanol-soluble material was dissolved in the above buffer solution and prepared for electrophoresis as described for the SDS-soluble fraction. The chloroform-methanol-insoluble fraction (the residue from the above extraction) also was dried under nitrogen gas, suspended in the sample buffer, and prepared for electrophoresis as described. Protein concentrations were determined by the method of Lowry et el. (9) using bovine serum albumin as a standard. SDS-gel electrophoresis was conducted as described by Maize1 (lo), with 8 x 0.6-cm, 13% polyacrylamide resolving gels and 0.5 x 0.6-cm, 4% spacer gels. Sample volumes were 10 to 100 ~1 and usually contained about 10,000 cpm; the quantities of protein applied to the gels are indicated in the figures. Electrophoresis was performed at 100 V (constant voltage) until the tracking dye was about 5 mm from the bottoms of the gels. A mechanical gel slicer was used to prepare the l-mm slices whose contents were then solubilized (11) and analyzed in a Nuclear Chicago 6848 liquid scintillation spectrometer. For molecular weight estimations the gels were stained according to Maize1 (10); molecular weight estimates of the mitochondrial proteins were calculated as described by Weber and Osborn (12) after electrophoresis of cytochrome c, chymotrypsinogen A, hen egg albumin, bovine serum albumin, aldolase, and catalase through 13 and 15% acrylamide gels. We used the molecular weight values of these proteins supplied by the vendor (Boehringer Mannheim). All the experiments described in this report were performed two or more times, and the examples cited represent typical results. RESULTS

In Table I is shown the distribution of protein and radioactivity in the mitochondrial fractions of spores labeled with P4Clleucine in the presence of cycloheximide in the interval between 240 and 300 min after the spores were placed in the germination medium and had reached

608

BRAMBL TABLE

AND

I

FRACTIONATION OF PROTEIN AND RADIOACTIVITY FROM MITOCHRONDRIA OF SPORES LABELED WITH [W]LEUCINE BETWEEN 240 AND 300 MIN OF GERMINATION IN THE PRESENCE OF CYCLOHEXIMIDE Total Total Fraction procounts tein per minute (mg) Whole mitochondria” Methanol extract of intact mitochondria

6.50 0.02

78,100 26,600

SDS-soluble extract SDS-insoluble residue

2.15 1.04

21,300 5,300

Neutral chloroform-methanol extract Neutral chloroform-methanol residue

0.40

8,000

2.47

16,600

n The mitochondrial fraction prepared from 990 mg (fresh weight) of spores was extracted with 90% methanol to remove unincorporated radioactivity and halved for the subsequent extractions with SDS or with chloroform-methanol, as described in the text.

93%. germination of maximum for substituted was P4Clleucine [3H]leucine in this particular experiment to minimize effects of sample self-absorption encountered in certain spore fractions. Intact mitochondria, prepared from labeled spores, were extracted with 90% methanol, a step which removed 34% of the total mitochondrial radioactivity and almost none of the protein. The methanolextracted mitochondria were then extracted with 1% SDS or with neutral chloroform-methanol. In the mitochondria labeled in the presence of cycloheximide, the SDS-soluble proteins contained approximately 83% of the total (postmethanol extraction) mitochondrial radioactivity, while the chloroform-methanol-soluble and -insoluble fractions contained about 31 and 65% of the total radioactivity, respectively. Approximately 66% of the total mitochondrial protein was soluble in SDS, and 12% of this total protein was soluble in neutral chloroform-methanol. In all the experiments described in this report, we extracted intact mitochondria rather than sonicated mitochondrial membranes or “submitochondrial particles” (4) because our initial experiments showed that, at

HANDSCHIN

some stages of spore germination, up to 50% of the cycloheximide-resistant protein synthesis products were lost upon disruption of the mitochondria by sonication. The [“Hlleucine-labeled mitochondrial proteins synthesized in the presence of cycloheximide between 240 and 300 min of germination were analyzed by electrophoresis through 13% polyacrylamide containing 0.1% SDS. Figure 1A shows the electrophoretic pattern of the radioactivity of the SDS-soluble proteins extracted from the mitochondria, and Figs. 1B and C show the patterns of the electrophoresed chloroform-methanol-soluble and -insoluble proteins, respectively. The highly reproducible gel radioactivity pattern of the SDS-extracted proteins shows that this fraction contained about eight to ten protein bands, some of which were not well resolved. In initial experiments, we tested the usefulness of the proteinase inhibitor phenylmethylsulfonyl fluoride to learn whether the observed patterns would be altered in the absence of possible proteolytic activity in the mitochondrial extracts; the results showed that presence of this inhibitor at several concentrations had no perceptible effect upon the electrophoretic patterns. The predominant peaks of radioactivity in the SDS extract of the mitochondria labeled in the presence of cycloheximide have apparent molecular weights of approximately 85,100, 56,900, 38,000, 33,100, 20,200, 15,300, 13,800 and 8400; the major portion of the SDS extract activity falls in the molecular weight region of 60,000 to 30,000 and a smaller amount between 20,000 and about 8000. These apparent molecular weight values are primarily useful for descriptive purposes and should not be regarded as absolute molecular weight determinations. Electrophoresis of the chloroform-methanol extract of the labeled mitochondria showed that only one protein, with a molecular weight of about 8400, was soluble in this solvent system. The chloroformmethanol-insoluble fraction appeared to contain most of the remainder of the proteins seen in the SDS-extracted fraction, the most prominent peaks having molecular weights of approximately 38,000 and

SPORE APPARENT

MOLECUIJIR

GEL SLICE

MITOCHONDRIAL WEIGHT x lO-3

NUMBER

FIG. 1. Electrophoresis of mitochondrial proteins synthesized by 240-300-min spores in the presence of cycloheximide. (A), SDS-soluble proteins, 150-pg sample; (B), chloroform-methanol-soluble protein, 150-pg sample; and (Cl, chloroform-methanol-insoluble proteins, loo-Kg sample. The apparent molecular weight values were determined as described in the text; migration is from left to right. The closed blocks indicate positions of the tracking dye. Background counts have been subtracted from the data in this and all subsequent figures. The one or two peaks of radioactivity observed in gel slices l-6 in

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SYNTHESIS

609

16,000. The results of this neutral chloroform-methanol extraction of the spore mitochondria agree with the results obtained with yeast (4) in that the chloroformmethanol extract in both cell types contains predominantly a single low molecular weight peptide, but the present results contrast with those observed with yeast in that the chloroform-methanol residue of the spores apparently contains most of the higher molecular weight peptides observed in the SDS extract. In the spore mitochondria, there is no apparent conversion of the higher molecular weight proteins into lower molecular weight bands under these neutral solvent extraction conditions. It was reported recently (8) that the isotope from 13Hlleucine (but not 13Hlphenylalanine) can be incorporated into a phospholipid-like fraction of cycloheximide-treated Neurosporu cells. In our experiments (data not shown) we established that the labeling of spore mitochondrial proteins with 13Hlphenylalanine gave results which were identical to those obtained with [3H]leucine. Furthermore, the 13Hllabeled radioactivity of the isolated 8400 molecular weight peptide was converted to free amino acid by acid hydrolysis, and the majority of the radioactivity extracted from the spore mitochondria by chloroform-methanol was solubilized after digestion with Pronase, with a corresponding diminution of radioactivity in the 8400 molecular weight peak in the gel pattern. The properties of the 8400 molecular weight peptide were examined in more detail. Electrophoresis of this chloroformmethanol-soluble protein through several different gels with acrylamide gel concentrations up to 20% did not separate this band into more components. If the intact mitochondria were extracted first with 5% trichloroacetic acid (as a substitute for 90% methanol), nearly all the higher molecular weight radioactivity seen in the SDS-extract gel patterns was converted to a form of about 8400 molecular weight. In Fig. 2A, 17,716 cpm of sample (100 pg of promost of the electrophoretic patterns are caused by accumulation of protein at the tops of the spacer and resolving gels.

610

BRAMBL

AND

tein) were applied to the gel in this experiment, and in Fig. 2B, 17,078 cpm of sample (100 pg of protein) were applied. The summed radioactivity (minus background counts) observed in the gel of Fig. 2A is 15,390 cpm and in the gel of Fig. 2B, 15,738 cpm. If the methanol-extracted mitochondria were transferred to a pH 10 buffer solution before SDS extraction, no such conversion of high to low molecular weight forms occurred. Because of the conversion of the higher molecular weight proteins to an apparent single molecular weight form after trichloroacetic acid treatment, it seemed possible that the 8400 molecular weight peptide

GEL

SLICE

NUMBER

FIG. 2. Electrophoresis of SDS-soluble proteins from mitochondria extracted first with 90% methanol (A) or with 5% trichloroacetic acid (B). The mitochondrial proteins were labeled in the presence of cycloheximide; each sample contained 100 pg of protein and about 17,000 cpm. The closed blocks indicate positions of the tracking dye.

HANDSCHIN

could have a precursory relationship to the other proteins seen in the SDS-soluble fraction. To examine this possibility, we measured the synthesis of these proteins at three points after initiation of cycloheximide inhibition in 300-min spores and addition of 13H]leucine. The results indicate that, after 20 min of labeling (Fig. 3A), nearly all the incorporated radioactivity was contained in the 8400 molecular weight peptide; after 40 min of labeling (Fig. 3B) the radioactivity appeared in the higher molecular weight proteins as well as the lower form. Finally, after 60 min of labeling (Fig. 3C), the radioactivity was distributed throughout all the peptides, and further incubation did not change this pattern. An examination of the chloroform-methanol-insoluble protein fractions of the spores incubated for these intervals confirmed that the 13Hlleucine appeared first in the higher molecular weight proteins only after 20 min and after synthesis of the 8400 molecular weight protein. Pulse-chase experiments in which the L3Hlleucine-labeled spores were incubated with unlabeled leucine both at 0 and 34°C gave results (data not shown) which also indicated that the low molecular weight protein was synthesized first and that the label subsequently was transferred to the higher molecular weight forms. The major object of this study was to measure the synthesis of mitochondrial proteins during spore germination to establish at what point the mitochondrial ribosomes become functional and whether patterns of mitochondrial protein synthesis change during the germination sequence as new mitochondria presumably are organized. The germination sequence of these spores was divided arbitrarily into five 60min periods for purposes of this study (see Discussion for a description of some of the metabolic events which occur during these periods). In Fig. 4 are shown the electrophoretic patterns of radioactivity of the chloroform-methanol-soluSDS-soluble, ble, and chloroform-methanol-insoluble proteins extracted from the spore mitochondria after labeling the spores with [3H]leucine in the presence of cycloheximide at five intervals of germination. It is evident that very little, if any, of

SPORE

MITOCHONDRIAL

the mitochondrial ribosome products was synthesized during the first 60 min of germination; no significant radioactivity could be detected by electrophoresis of any of these fractions from the 60-min spores.3 During the second interval of germination (60-120 min), synthesis of cycloheximideinsensitive ribosome products in the mitochondria was readily observed. As shown in the SDS-soluble protein electrophoretic pattern, the major product was the 8400 molecular weight peptide which is soluble in chloroform-methanol. The higher molecular weight regions of the gel contained only small amounts of labeled protein; the relatively small amount of chloroformmethanol-insoluble material present electrophoresed close to the 8400 molecular weight peptide. The mitochondrial ribosome products synthesized during the third interval (120-180 min) showed a generally similar electrophoretic pattern, except for the presence of somewhat increased quantities of the higher molecular weight proteins seen in electrophoretic patterns of both the SDS-soluble and the chloroform-methanol-insoluble fractions. The 8400 molecular weight peptide remains the predominant protein synthesized in this interval, however. In the last two intervals of inhibition and labeling (MO-240 and 240-300 min), the high molecular weight proteins were synthesized in much larger proportion to the 8400 molecular weight peptide, as shown by electrophoresis of the SDS-soluble mitochondrial proteins. Furthermore, the specific activities of the chloroformmethanol-insoluble mitochondrial proteins were increased in relation to the earlier periods, and the electrophoretic patterns of the chloroform-methanol-insoluble protein contained a larger quantity of high molecular weight proteins. These distinctive, germination stage-specific patterns of synthesis of mitochondrial proteins indicate that the mitochondrial ribosomes do not synthesize qualitatively and quantita’ If this first interval (O-60 min) of incubation in cycloheximide and [3H]leucine was extended to 30 min, it was possible to observe synthesis of mitochondrial ribosome products which otherwise appeared in the second (60-120 min) interval.

PROTEIN

SYNTHESIS

611

800

A

n

20 mu7

7cQ600 500 400300 200. 100

m if 2

zi

600 oi;;I

B

40 mm

500

i r

400 -

5 8

300 200'

100 0. ..wi:

0

I

20

40

60

SO

GEL SLICE NUMBER 3. Electrophoresis of SDS-soluble mitochondrial proteins synthesized in the presence of cycloheximide 20 min (A), 40 min (B), and 60 min (Cl after addition of [UHlleucine. All samples contained 100 pg of protein. The closed blocks indicate positions of the tracking dye. FIG.

tively identical products during the 300min germination sequence. For purposes of comparison, the synthesis of mitochondrial proteins in the ab-

612

BRAMBL

AND

HANDSCHIN

60.t2Omm C M-INSOLUBLE

i 200 r

200

I

t

t20-lBOm,n SDS- SOLUBLE /25O//gJ

izo-l8Omm c. M-SOLUeLEPOO~qJ n

tao 240 /n,n SDS SOLUEL E /25O,ugJ

1

/3OOpqJ

I20 -lBOmm C,Lf- INSOLUBLE /3OO.uq/

I

I 1

240.3OOmn SDS- SOLUBLE ft5O,,qJ

iEiz%zE

/2OOpqJ

240.3OOmm c:kf SOLUBL E /l5OpqJ c

240-3OOmm CM-lNSOLUELE/lOOpq/

GEL SLICE NUMBER FIG. 4. Electrophoresis form-methanol-insoluble intervals of germination. each panel. The ordinate the tracking dye.

of SDS-soluble, proteins labeled The quantities of scale is the same

chloroform-methanol (C:M)-soluble, in the presence of cycloheximide at protein applied to the gel columns are in all plots. The closed blocks indicate

and chlorofive 60-min indicated in positions of

SPORE

MITOCHONDRIAL

sence of cycloheximide inhibition was measured. These results would be expetted to show the contribution of the cytoplasmic ribosomes (as well as the mitochondrial ribosomes) to the labeled proteins associated with mitochondria. Shown in Fig. 5 are the electrophoretic patterns of extracted mitochondrial proteins (synthesized in the absence of inhibition) of only the first and last intervals of germination (O-60 and 240-300 min). A comparison of these results with the counterpart patterns of Fig. 4 shows that cycloheximide inhibition caused the disappearance of severa1 high molecular weight proteins and a reduction of radioactivity in certain other electrophoretic bands. It is noteworthy that products of the cytoplasmic ribosomes became associated with the mitochondria during the first 60 min of germination when the mitochondrial ribosomes were not functional. It also should be noted that in the absence of cycloheximide, as in its

PROTEIN

613

SYNTHESIS

presence, none of the 8400 molecular weight chloroform-methanol-soluble protein was synthesized during the first 60 min of germination, and the proteins seen in the SDS extract are similar, if not identical, to those found in the chloroformmethanol-insoluble fraction. In the 240300 min interval, the only chloroformmethanol-soluble protein synthesized in the absence of cycloheximide inhibition is the 8400 molecular weight protein synthesized in the presence of the drug. The results presented to this point provide indirect evidence that the mitochondrial proteins synthesized in the presence of cycloheximide are mitochondrial ribosome products. If these proteins are indeed products of the organelle ribosomes, their synthesis should be abolished by chloramphenicol, and Fig. 6 shows that this is the case. The peptides that had been labeled and extracted from mitochondria of cycloheximide-inhibited spores were not seen in o-60mn L M- /NSOUJEL E /25j,gj

600

400

j0

200

0

0

-I

100 n

GEL SLICE

240-300 rn,” C M-INSOLUBLE l25pgJ

NUMBER

FIG. 5. Electrophoresis of SDS-soluble, chloroform-methanol (C:M)-soluble, and chloroform-methanol-insoluble mitochondrial proteins synthesized in the absence of cycloheximide at two intervals of germination (O-60 and 240-300 min). The quantities of protein applied to the gel columns are indicated in each figure. The closed blocks indicate positions of the tracking dye.

614

BRAMBL

AND

600

500

400

300

200

100 P 1 f is F

O 200

loo

C

600

500

400 300

200

100

0

I 0

20

40

GEL SLICE

60

80

NUMBER

FIG. 6. Electrophoresis of SDS-soluble (A), chloroform-methanol-soluble (B), and chloroformmethanol-insoluble (C) mitochondrial proteins labeled in the presence of chloramphenicol between 240 and 300 min of germination. Gel samples (A) and (C) each contained 25 pg of protein and (B) contained 100 pg. The closed blocks indicate positions of the tracking dye.

the gel patterns of mitochondrial proteins from chloramphenicol-inhibited spores. DISCUSSION

In the physiologically mature, germinated spores, we have observed that about eight to ten proteins are synthesized in the presence of cycloheximide and are extract-

HANDSCHIN

able from mitochondria with SDS. The synthesis of these mitochondrial proteins, whose apparent molecular weights range from 85,100 to approximately 8400, is inhibited by chloramphenicol, an inhibitor of mitochondrial ribosomes. Our results show that the mitochondrial ribosomes are not functional during the first 60 min of germination, although, during this same period, proteins synthesized on cytoplasmic ribosomes are incorporated into or associated with the mitochondria. These cytoplasmic ribosome products must be coded for by the latent messenger RNA which we have shown previously to be present in the dormant spores and translated upon the onset of germination (2). Cytoplasmic ribosome activity appears not to be required for initiation of mitochondrial ribosome activity since incubation of spores for 90 min with cycloheximide will permit synthesis of mitochondrial ribosome products. The smallest mitochondrial ribosome product, the 8400 molecular weight peptide, was soluble in neutral chloroformmethanol, while the other, higher molecular weight products of the mitochondrial and cytoplasmic ribosomes were insoluble in this solvent. When the mitochondria were treated with trichloroacetic acid before SDS extraction of the labeled proteins, subsequent electrophoresis showed that the higher molecular weight peptides were converted to a single band in the 8400 molecular weight region of the gel. Tzagoloff and Akai (4) observed similar effects after extraction of yeast mitochondria with chloroform-methanol, and Ktintzel et al. (8) recently showed that performic acid oxidaproteins tion of Neurospora mitochondrial resulted in an accumulation of low molecular weight peptides at the apparent expense of the higher molecular weight products of mitochondrial ribosomes. In contrast to the results obtained with the yeast mitochondria (41, the chloroform-methanol extraction does not disrupt the higher molecular weight proteins of our spore mitochondria as shown by electrophoresis of the chloroform-methanol-insoluble protein fraction (Fig. 1). The functional role of the mitochondrial

SPORE

MITOCHONDRIAL

translation products in general and the low molecular weight hydrophobic proteins in particular (such as the 8400 molecular weight peptide) is not established. In yeast (4) and Neurospora (8) a hydrophobic peptide of about 8000 molecular weight appears to be a major product of the mitochondrial ribosomes. This peptide is associated in some way with higher molecular weight proteins synthesized in the presence of cycloheximide; in both organisms the latter proteins can be disrupted to yield the low molecular weight peptide. In Neurospora the lower molecular weight proteins are synthesized either first or at a faster rate after initiation of cycloheximide inhibition (14). We have observed both effects in our spore mitochondria. Furthermore, in yeast it has been shown (15) that a hydrophobic product of the mitochondrial ribosomes (with a molecular weight of about 7800) seen in chloroformmethanol extracts of mitochondria labeled in the presence of cycloheximide probably is identical to the subunit 9 of the rutamyadenosine triphosphatase tin-sensitive complex. From our experiments, we suggest that the 8400 molecular weight peptide is a precursor or a component of the other higher molecular weight proteins observed by electrophoresis of mitochondrial extracts. This peptide is the first to be synthesized after addition of 13Hlleucine to spores incubated in the presence of cycloheximide, and pulse-chase labeling experiments indicate a transfer of radioactivity from the lower to the higher molecular weight proteins. It seems possible that early in spore germination the mitochondrial ribosomes synthesize the 8400 molecular weight peptide which may serve as a precursor to the other mitochondrial proteins assembled subsequently. Alternatively, this peptide may become associated with unlabeled cytoplasmic peptides (synthesized before cycloheximide inhibition and incorporated into the mitochondria) to yield the distribution of acid-labile, higher molecular weight, [3Hllabeled proteins composed of unlabeled cytoplasmic ribosome products of different molecular weights and a i3Hl labeled mitochondrial ribosome product of

PROTEIN

SYNTHESIS

615

constant molecular weight. There is no evidence available now to favor either hypothesis, and the function of this low molecular weight peptide in the elaboration of the respiratory system of germinating spores is being examined further. The indication that all products of the mitochondrial ribosomes are not synthesized simultaneously in the physiologically mature, germinated spores is important in interpreting the transient differences in synthesis of mitochondrial ribosome products during several intervals of germination. The first products of the mitochondrial ribosomes were synthesized between 60 and 120 min of germination; these earliest products were primarily the chloroform-methanol-soluble 8400 molecular weight peptide and a smaller quantity of slightly higher molecular weight proteins which were insoluble in this solvent. During the next interval (120-180 min), this basic pattern was repeated, although somewhat more radioactivity appeared in the higher molecular weight chloroformmethanol-insoluble peptides. After 180 min of germination, the electrophoretic patterns of the extracted mitochondrial proteins changed dramatically, however; and the mitochondria were first seen to contain the full complement of labeled mitochondrial peptides characteristic of the germinated spores or mycelial cells. After 180 min the SDS extract contained large amounts of the higher molecular weight proteins as well as the 8400 molecular weight peptide. These findings are consistent with a recent report by Hawley and Greenawalt (13) which described electrophoresis of whole cell extracts of Neurospora conidia labeled in the presence of cycloheximide; in the 8-h cells they observed synthesis of about 10 proteins. Although the extraction and analysis methods used by these authors were somewhat different from ours, it is noteworthy that they observed early synthesis (O-60 min) of low molecular weight proteins which quantitatively diminish in proportion to higher molecular weight proteins as the spores germinate. The synthesis of these mitochondrial ribosome products perhaps is significant in

616

BRAMBL

AND

relation to several other metabolic activities of these spores. We have shown previously (1, 2) that nuclear RNA synthesis is begun at high rates between 90 and 120 min of germination, followed by a sharp increase in the rate of cytoplasmic protein synthesis. Ethidium bromide-sensitive mitochondrial DNA synthesis is begun between 120 and 180 min of germination (31, and an accelerated phase of cyanide-sensitive spore oxygen consumption is begun at about 150 min (1,3). Cytochrome synthesis appears to be initiated at about 180 min (R. Brambl and M. Josephson, in preparation), and cyanide-sensitive cytochrome c oxidase activity cannot be detected in the spore mitochondria until about 150 min (R. Brambl, unpublished data). Finally the spore germ tubes begin emergence at about 150 min provided the cytoplasmic ribosomes have been functional at least through the first 135 min of germination (1). Our previous studies with chemical inhibitors (1, 3) have shown that function of the mitochondrial genetic system is not required for spore respiration (in the first 150 min of incubation) or for germination, although function of the cytoplasmic ribosomes is essential. The results of the present study show directly that the mitochondrial ribosomes do not function during the first 60 min of germination and that the mitochondria do not develop the full complement of mitochondrial ribosome products until 120-180 min of germination. The present results support our earlier suggestion (1) that these dormant spores contain a preserved, potentially functional mitochondrial aerobic respiratory system which requires a contribution from the cytoplasmic ribosomes to become active in the absence of activity of the mitochondrial genetic system. Function of the mitochondrial ribosomes is not involved in this initial activation of the respiratory system, although products of these ribosomes are required for subsequent organization of new mitochondria around 150 min of germination. We previously reported results of respiration experiments whose measurements were made following incubation of these spores in several sequential combinations

HANDSCHIN

of cycloheximide and chloramphenicol (1). These results suggested that if the cytoplasmic ribosomes were inhibited early in germination (through 180 min), a mitochondrial ribosome product could accumulate. After reversal of the ribosome inhibition in the second phase of the experiment, these accumulated mitochondrial ribosome products could then combine with newly synthesized cytoplasmic products to permit development of mitochondrial respiration. These interpretations predicted the independent synthesis early in germination (before 180 min) of a mitochondrial ribosome product in the absence of cytoplasmic ribosome activity. Results presented in this present report support this earlier prediction, and it seems most likely to us that the 8400 molecular weight peptide is the mitochondrial ribosome product which is synthesized in the absence of cytoplasmic ribosome function, which accumulates unused, and which later permits the emergence of mitochondrial respiration as the cytoplasmic ribosomes are relieved from cycloheximide inhibition. ACKNOWLEDGMENTS This research was supported in part by a Faculty Grant-in-Aid of Research from the University of Minnesota Graduate School and by NIH Research Grant GM-19398 from the National Institute of General Medical Sciences. REFERENCES 1. BRAMBL, R. (1975) Biochim. Biophys. Acta 396, 175. 2. BRAMBL, R. M., AND VAN ETTEN, J. L. (1970) Arch. Biochem. Biophys. 137, 442. 3. DUNRLE, L. D., VAN ETTEN, J. L., AND BRAMBL, R. (1972) Arch. Mikrobiol. 85, 225. 4. TZAGOLCJFF, A., AND AKAI, A. (1972) J. Biol. Chem. 247, 6517. 5. ROGERS, P. J., AND STEWART, P. R. (1974) J. &zcterioZ. 119, 653. 6. LANSMAN, R. A., ROWE, M. J., AND WOODWARD, D. 0. (19741Eur. J. Biochem. 41, 15. 7. ROWE, M. J., LANSMAN, R. A., AND WOODWARD, D. 0. (1974)Eur. J. Biochem. 41, 25. 8. K~NTZEL, H., PIENIA~EK, N. J., PIENIAZEK, D., AND LEISTER, D. E. (1975)Eur. J. Biochem. 54, 567. 9. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., ANDRANDALL, R. J. (1951) J. Biol. Chem. 193, 265. 10. MAIZEL, J. V. (1971) in Methods in Virology

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(Maramorosch, K., and Koprowski, H., eds.), Vol. 5, p. 179, Academic Press, New York. 11. ZAITLIN, M., AND HARIHARASUBRAMANIAN, V. (1970) And. Biochem. 35, 296. 12. WEBER, K., AND OSBORN, M. (1969) J. Biol. Chem. 244, 4406.

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13. HAWLEY, E. S., AND GREENAWALT, J. W. (1975) Eur. J. Biochem. 54, 585. 14. MICHEL, R., AND NEUPERT, W. (1973) Eur. J. Biochem. 36, 53. 15. TZAGOLOFF, A., RUBIN, M. S., AND SIERRA, M. F. (1973) Biochim. Biophys. Acta 301, 71.

Mitochondrial biogenesis during fungal spore germination: products of mitochondrial protein synthesis in vivo.

ARCHIVES OF BIOCHEMISTRY Mitochondrial AND BIOPHYSICS 175, 606-617 (1976) Biogenesis during Fungal Spore Germination: of Mitochondrial Protein...
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