Protease from Chiamydomonas reinhardtii'

Plant Physiol. (1992) 99, 932-937 0032-0889/92/99/0932/06/$01 .00/0

Received for publication December 9, 1991 Accepted February 6, 1992

Purification and Characterization of a Membrane-Bound Protease from Chiamydomonas reinhardtii' J. Hughes Department of Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania 19140 J.

Kenneth Hoober*2 and Marie

ABSTRACT In Chiamydomonas reinhardtii y-1, newly synthesized chlorophyll a/b-binding apoproteins are degraded when chlorophylls are not present for assembly of stable light-harvesting complexes. A protease was purified from the membrane fraction of degreened y-1 cells, which digested chlorophyll a/b-binding proteins in membranes from C. reinhardtii pg-1 13, a protease-deficient strain. This protease was active with p-nitroanilides of nonpolar amino acids (Leu and Phe), but not of basic amino acids (Lys and Arg). The apparent molecular weight of the enzyme is 38,000 ± 2,000 as determined by electrophoresis in the presence of sodium dodecyl sulfate. Typical inhibitors of the major classes of proteases were ineffective with this enzyme. Protease activity was constant from pH 7.5 to 9; a plot of log V versus pH suggested that deprotonation of an ionizable group with a pK value of 6.0 to 6.5 is required for activity. The protease was inactivated by diethylpyrocarbonate and by photooxidation sensitized by rose bengal. These results suggested that a histidyl residue is required for catalysis. Although very sensitive to photodynamic conditions in vitro, the enzyme was not inactivated in vivo when cells were exposed to light.

the Cab proteins that accumulate is illustrated by the minimal digestion that occurs when exogenous proteases are added to membranes from these cells. In contrast, cells of the pg-1 13 strain of C reinhardtii accumulate amounts of Cab proteins typical of green cells (23) even though they are unable to synthesize Chl b (7) and, thus, do not assemble Chl a/bprotein complexes. Cab proteins are associated with membranes in pg-1 13 cells, yet are extensively digested by exogenous proteases (12). The increased sensitivity of Cab proteins in pg-1 13 cells suggests that, without Chl b, folding of these proteins remains incomplete. Thus, it is possible that the pg113 cells are deficient in an important protease that normally would degrade the incompletely folded Cab proteins. A comparison of protease activities in pg-113 cells with those in y-l cells indicated a marked difference in a membrane-bound protease (12). In this article, we describe the purification of this protease from membranes of y-1 cells and the characterization of some of its properties, in particular its sensitivity to various inhibitors. MATERIALS AND METHODS

Cells

A family of stable, Chl a/b-protein complexes serves as the major light-harvesting antennae in thylakoid membranes (2, 26). Cytoplasmically made Cab3 proteins do not generally accumulate unless Chl are synthesized in the chloroplast at the same time (10, 12). The available evidence suggests that Cab apoproteins are rapidly degraded by proteases after they enter the chloroplast (3, 10, 12, 28). During chloroplast development, however, the ensuing stability of Cab proteins may result from regulation of protease activity and/or an ability of Chl to initiate folding of the proteins into proteaseresistant Chl-protein complexes. To understand these processes, it is necessary to identify and characterize proteases involved in degradation and also elucidate the mechanisms involved in assembly of stable Chl-protein complexes. Cells of the y-1 strain of Chlamydomonas reinhardtii express the cab genes at 380C but are unable to synthesize Chl in the dark (13). Consequently, accumulation of Cab proteins is minimal until cells are exposed to light (12). The stability of

Chlamydomonas reinhardtii y-1 cells were grown in the dark to deplete the chloroplast of thylakoid membranes as described previously (12, 21). Cells were harvested, the packed cells were suspended in 5 volumes of 50 mm Tricine-NaOH, pH 8.0, containing 1 mM MgCl2 (buffer A), and the suspension was passed through a French pressure cell at 6,000 p.s.i. The broken-cell sample was centrifuged for 1 min at 2,000g to sediment remaining whole cells and then at 30,000g for 30 min at 50C. The membrane pellet was resuspended in buffer A and repelleted.

Purification of the Protease The crude membrane fraction was suspended in 3 volumes of buffer A and added dropwise to sufficient acetone, prechilled to -200C, to provide a final acetone concentration of 92% (v/v). This step extracted lipid and pigments and allowed subsequent solubilization of the enzyme. Acetone-insoluble protein was collected by centrifugation and dried under nitrogen. Protease was extracted from this material with 0.8 M ammonium sulfate (20% saturation) and then precipitated at 3 M ammonium sulfate (75% saturation). The 3 M ammonium sulfate-insoluble fraction was suspended in buffer A, clarified by centrifugation, and desalted on a column (2.5 x 35 cm) of Sephadex G-25 equilibrated with 5 mm NaPi, pH 7.5. Frac-

1 This work was supported by National Science Foundation grants DCB-8613585 and DCB-9018797. 2 Present address: Department of Botany, Arizona State University, Tempe, AZ 85287-1601. 3Abbreviations: Cab, Chl a/b-binding protein; CMPS, p-chloromercuriphenyl sulfonate; DEPC, diethylpyrocarbonate; NA, p-nitroaniline or p-nitroanilide; RB, rose bengal.

932

l~ ~ ~ ~ ~ ~ ~

MEMBRANE PROTEASE FROM CHLAMYDOMONAS

tions containing protease activity were applied to a column (1.5 x 15 cm) of DEAE-Sephadex A-50 and eluted with a 200-mL gradient of 0 to 0.25 M NaCl in 10 mm NaPi, pH 7.5, at a flow rate of 0.24 mL/min. The fractions containing activity (about 12 mL total volume) were diluted threefold with water, applied to a smaller column (0.8 x 6 cm) of DEAE-Sephadex A-50, and eluted with 10 mm NaPi, pH 7.5, containing 0.2 M NaCl to concentrate the protease.

A

_7

E z -5

0

E

i'_I

Assays 000

Purification of Chlamydomonas Protease Sucrose gradient analysis of membranes from y-1 cells indicated that the protease resided in a membrane with a density slightly lower than that of thylakoid membranes (12). Included in membranes of this density are those of the chloroplast envelope. The specific activities of the protease (nmol NA produced/min * mg protein) in the membrane fraction from degreened cells or from fully green cells were similar, which indicated that the activity increased along with development of thylakoid membranes. However, we wanted to focus on the activity present at the time degreened cells were initially exposed to light. Therefore, y-l cells were degreened to reduce the amount of thylakoids in the membrane fraction prior to purification. Table I summarizes preparation of the partially purified protease with which most of the characterization studies were performed. This preparation, which was purified over 200fold from the membrane fraction, contained several polypeptides resolved by SDS-PAGE (Fig. lb). The major components were polypeptides of Mr 38,000 and 36,000. Attempts to

Table I. Partial Purification of Protease from Cells of C. reinhardtii y- I The data were obtained with 1 x 1010 cells from a 2.5-L culture. Fraction

Total Activity Protein Specific Activity nmol Leu-NA Atmol Leu-NA mg hydrolyzed/ hydrolyzed/minl min

Homogenate Crude membranes Acetone pellet 3 M (NH4)2SO4 precipitate DEAE-Sephadex eluate

19.6 7.6 6.9 4.4 4.0

mg

289

68

114

67

112 11 0.29

62 400 14,000

.-

0

.

14-0-*

-WOOOO**

r__ w

Fraction b

c

BSA- gt>i~

-BSA -CA

STIC yt crr

RESULTS

Cyt c

,3 r.

;

Protease activity was routinely assayed at 250C in 50 mm Tricine-NaOH, pH 8.0. Release of NA from amino acyl- and peptidyl-p-nitroanilides was monitored continuously at 405 nm (27, 29). Protein was measured with the bicinchoninic acid reagent as described by the supplier (Pierce Chemical Co., Rockford, IL) or the procedure of Bradford (4), with BSA as the standard. Chl was measured in 80% acetone extracts (1). SDS-PAGE was performed as described previously (14). Electrophoresis in nondenaturing gels was done as described by Moriyasu et al. (25). Isoelectric focusing was performed in free solution with the Rotofor cell (Bio-Rad).

CA v

BSA V7

E

933

- STI

-Cytc

Figure 1. Analysis of mol wt of the Chlamydomonas protease. a, Elution pattern of protease, purified as in Table I, from an additional Sephacryl S-200 column (0.9 x 32 cm) in buffer A. b, SDS-PAGE of a protease preparation purified through the DEAE-Sephadex step, as in Table I. c, SDS-PAGE of protein in fractions 10 through 20 (each 1.2 mL), collected from the S-200 column as shown in a. Mr 38,000 and Mr 36,000 polypeptides were present in fractions that contained protease activity. Artifactual bands appeared in all fractions near the position of BSA. The positions of marker proteins are indicated. BSA (Mr 66,300); CA, carbonic anhydrase (Mr 29,000); STI, soybean trypsin inhibitor (Mr 20,100); Cyt c, horse heart Cyt c

(Mr 12,400). further purify the enzyme included introduction of a column (1.5 x 60 cm) of Sephacryl S-200 equilibrated with 5 mm NaPi, pH 7.5, before the DEAE-Sephadex step in place of the Sephadex G-25 column. After the two DEAE-Sephadex columns, the active fractions were passed through a second Sephacryl S-200 column (0.9 x 32 cm) equilibrated with buffer A. This procedure was developed because the protease eluted as a protein of apparent mol wt 75,000 ± 5,000 from Sephacryl S-200 in 5 mm NaPi, pH 7.5, but with an apparent mol wt of 47,000 ± 3,000 from the same column equilibrated with the Tricine buffer (Fig. la). The elution behavior suggested that the protease has a tendency to aggregate, a conclusion supported by its low mobility during electrophoresis in nondenaturing gels (not shown). The isoelectric point of the protease is 6.0. The active fractions from the final Sephacryl S-200 column (Fig. la) contained a major polypeptide of Mr 36,000 ± 2,000 and a minor polypeptide of Mr 38,000 ± 2,000 (Fig. ic). The specific activities of several such preparations were variable

934

HOOBER AND HUGHES

Plant Physiol. Vol. 99, 1992

(6), which as shown in Figure 2 was not achieved in the low pH range, the derived pK value remains an approximation.

0~~~~~~

0>40

20

0I

4

5

6

7

8

9

pH Figure 2. Effect of pH on observed rate of Leu-NA hydrolysis. The assayed in a series of 50 mm NaPi buffers at (0) 0.25, (U) 0.5, and (0) 1.0 mm Leu-NA. Slopes of curves for highest rates in the low and high pH ranges were extrapolated to provide an protease was

intersection at pH 6.2.

and lower than the reproducible values shown in Table I. Because the amount of the Mr 38,000 component in each fraction corresponded more closely to activity than the Mr 36,000 polypeptide, possibly the larger polypeptide was the active enzyme and the lowered specific activity resulted from autodigestion.

Characteristics of the Partially Purified Protease Substrate Specificity

The protease was highly active with Leu-NA and Phe-NA substrates. The Km values for Leu-NA and Phe-NA at 250C and pH 8.0 were 45 ± 3 and 21 ± 2 jAM, respectively, which indicated a high affinity of the enzyme for nonpolar amino acids. Maximal activity with Ala-NA was approximately 10% of that with Leu-NA. No activity was detected with Arg-NA or Lys-NA, or with the peptide substrates N-succinyl-PheNA, N-succinyl-Ala-Ala-Pro-Phe-NA, or N-succinyl-AlaAla-Pro-Leu-NA. as

Activity versus pH

The protease was maximally active between pH 7.5 and 9 and decreased in activity below pH 7. As shown in Figure 2, the enzyme probably was not saturated with substrate at low pH even at 1 mm Leu-NA. At higher pH values, a high substrate concentration decreased activity. Extrapolation of the slopes of the plot shown in Figure 2 provided an intersection at a pH value near 6.2, an apparent pK value for an ionizable group required for enzymic activity (6). Because determination of the pK value from this type of plot depends upon saturation of the enzyme with substrate at all pH values

Inhibitors The effects of a variety of typical inhibitors of proteases were determined with the Chlamydomonas protease. Inhibitors of serine-type proteases such as 3,4-dichloroisocoumarin (0.5 mM), leupeptin (1 mM), or aminobenzamidine (5 mM) had no effect on the enzyme activity. Addition of PMSF resulted in an immediate, concentration-dependent inhibition. In the presence of 1 mm PMSF, the protease was inhibited about 50%, but with time the inhibition gradually diminished. The decrease in inhibition corresponded approximately to the rate of hydrolysis of PMSF in water at pH 8.0 (15). The product of hydrolysis, phenylmethylsulfonate, was not inhibitory. In control experiments, inhibition of trypsin showed a gradual onset of loss of activity after PMSF was added until complete inhibition was obtained after 5 to 10 min of incubation, as expected for irreversible inhibition. These results suggested that PMSF did not inhibit the Chlamydomonas protease by forming a covalent adduct with an active-site serine. Several inhibitors of cysteine-type proteases such as Ltrans-epoxysuccinyl-1-leucylamido-(4-guanidino)butane (0.5 mM), antipain (0.35 mM), HgCl2 (0.1 mM), iodoacetate (20 mM), or methyl methane thiosulfonate (100 mM) also did not affect activity of the Chlamydomonas protease. However, 0.5 mM CMPS caused 50% inhibition at a substrate (Leu-NA) concentration of 1 mm. Inhibition by CMPS was dependent upon substrate, with the extent of inhibition increasing as the substrate concentration was raised. The extent of inhibition did not increase with time as would be expected for formation of an adduct with a sulfhydryl group. No inhibition occurred when the enzyme and inhibitor were preincubated 10 min before dilution into the assay mixture containing substrate. Because other potent inhibitors of cysteine-type proteases were not effective, we concluded that inhibition by CMPS was not the result of reaction with a sulfhydryl group. Addition of DTT to 1 mm in the assay mixture did not affect activity.

Metalloprotease inhibitors such as 1,10-phenanthroline (2 mM), EDTA (1 mM), or leucine hydroxamate (1 mM) did not inhibit the enzyme. Pepstatin A (0.8 mM), an inhibitor of aspartate-type proteases, also did not inhibit the Chlamydomonas protease.

Photodynamic Inactivation The Chlamydomonas protease was particularly sensitive to photodynamic inactivation in the presence of RB. To minimize extraneous photochemical effects, for most experiments samples were exposed to green fluorescent light, which has an emission maximum (530 nm) near the absorption maximum of RB (550 nm). Figure 3 shows inactivation as a function of RB concentration. At the higher concentrations, RB also inhibited the enzyme in the dark, which suggested direct interaction of the dye with the enzyme. Inhibition in the dark was slight at 0.1 gM RB, yet extensive inactivation occurred when the sample was exposed to green light. Addition of Leu-NA during the light exposure partially protected the enzyme from inactivation (not shown).

MEMBRANE PROTEASE FROM CHLAMYDOMONAS

935

to 83% of the control value. These results are consistent with involvement of a histidyl residue(s) in catalytic activity (24).

The Protease Is Not Photoinactivated in Vivo O 60

a) 40

20 20

0

1

2

3

4

5

Rose Bengal (,uM) Figure 3. Effect of RB concentration on the rate of Leu-NA hydrolysis. Protease was incubated at room temperature in 50 mm TricineNaOH, pH 8.0, for 5 min with various concentrations of dye in the dark (0) or in 5 Wm-2 green fluorescent light (Westinghouse Fl 5T8/ G) (0), and then substrate was added to assay for activity. Fluence of light was measured with a Yellow Springs radiometer (model 65A).

Figure 4 shows rates of inactivation at several concentrations of RB. The half-life of protease activity at a light fluence of 5 Wm-2 was about 1 min in the presence of 1 ,UM RB and 4 min in 0.1 uM RB. When these experiments were repeated with proteolytic activities in the soluble fraction of the cells, no inhibition of Ala-NA or Leu-NA hydrolysis was observed at 1 $M RB (not shown). Methylene blue, which like RB is an effective generator of singlet oxygen (11, 17), at 5 gM did not significantly decrease activity of the protease in white light. Neutral red mediates radical-type photooxidation reactions (11), but also did not sensitize inhibition of the enzyme at 5 jAM in white light.

In experiments with Triton-solubilized crude membranes, photoinactivation of the protease required approximately 10fold higher concentrations of RB for the same decrease in activity observed with the purified enzyme. The requirement for higher dye concentrations probably was due to carotenoids and other antioxidants in the membranes. To test whether these higher concentrations of dye would affect this activity in vivo, cells were preincubated for 1 h at 380C in the dark and then RB was added to various concentrations. After an additional incubation for 15 min in the dark, cells were exposed to 5 Wm-2 green fluorescent light. After various periods of exposure to light, cells were collected, broken, and fractionated into soluble and membrane fractions. The membrane fraction prepared from these cells showed no loss of protease activity over 15 min of illumination, even when cells were incubated in 10 ZlM RB. At the higher concentrations of dye, washed cell pellets and membrane fractions were pink, which suggested that the dye was taken up by the cells.

c

E

E a) .S-

Inactivation by DEPC An apparent pK value near 6 (Fig. 2) and the sensitivity to photoinactivation with RB suggested that the protease contains an essential histidyl residue. To test this possibility further, the enzyme was treated at pH 8.0 and room temperature with DEPC, which generates N-carbethoxyhistidyl derivates in proteins. The protease was rapidly, but only partially inactivated with 1 mm DEPC at pH 8.0. Because DEPC has a half-life of about 30 s at pH 8.0 (24), four successive additions of DEPC, 2 min apart to a concentration of 1 mm each time, were made to the assay mixture. This treatment resulted in complete inactivation of the enzyme. Inactivation also occurred at pH 6.0, a condition more selective for derivatization of histidyl residues (24). After 1 h of incubation with 2 mm DEPC at pH 6.0, 17% of the activity remained. When the enzyme treated with DEPC at pH 6.0 was further incubated for 1 h with 0.1 M NH20H, activity was restored

c a)

0-a)

M inu tes Figure 4. Rates of photoinactivation of protease by RB. The protease was incubated at room temperature in 50 mm tricine, pH 8.0, with (0) 0.1, (0) 0.2, (C) 0.5, and (-) 1.0 gM RB in 5 Wm-2 green fluorescent light for the times indicated. Leu-NA was then added and remaining activity was measured.

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HOOBER AND HUGHES

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p

1992

Activation by Alcohols The protease, solubilized from the membrane fraction with 1% Triton X-100, was stimulated about 2.5-fold by alcohols. The greatest effect of alcohols occurred at high (1.0-1.5 mm) substrate concentrations. As shown in Figure 6, although maximal stimulation by each alcohol was similar, the concentration at which maximal stimulation was achieved varied according to chain length. Hydrolysis of Leu-NA by proteases in the soluble fraction was not affected by 10% isopropanol under these conditions.

2 3 45 1-

Physiol. Vol. 99,

' o_ _

DISCUSSION

II7

Figure 5. Digestion of Cab proteins in membranes from pg-i 13 cells by the protease. A strain of C. reinhardtii pg-i 13 that becomes yellow in the dark was grown for 3 d in the dark, suspended in fresh medium, incubated 1 h at 380C, and then exposed to light for 1 h (12). Membranes (100 Asg protein) prepared from these greening cells were incubated with purified protease (0.8 jIg protein) in 10 mm NaPi, pH 7.5, at 25C. Lanes 1 to 3, 0, 60, and 90 min of incubation, respectively, with protease. Lanes 4 and 5, 0 and 90 min of control incubation, respectively, with buffer. After SDS-PAGE, the gel was stained with Coomnassie blue (14). The positions of the major Chiamydomonas Cab proteins, polypeptides 11, 16, and 17, are indicated. A polypeptide remaining near the position of polypeptide 1 1 in lane 3 was probably polypeptide 12, which was shown earlier (12) to be protease resistant.

Similar results were obtained when cells were exposed to white light. Thus, antioxidants or a low 02 concentration possibly protected the protease from inactivation in vivo. These results indicate that accumulation of Cab proteins in y-l cells is not related to inactivation of the protease during

In C reinhardtii y-i cells, Cab proteins are apparently degraded upon import into the chloroplast unless protected by immediate interaction with Chl (12). We began a study of the proteases that potentially contribute to degradation of these proteins. The soluble fraction of C reinhardtii y-l contains a number of proteolytic activities (18, 22). Among these is a specific protease derived from the chloroplast stroma that removes the transit sequence from precursors of the Cab proteins (22). Further degradation of Cab proteins by the soluble fraction in vitro was minimal. However, if precursors of Cab proteins were incubated with membrane fractions from these cells, extensive degradation occurred (22). This observation, and the difference in activity of a membranebound protease between y-1 cells and those of the strain pg113 of C. reinhardtii reported previously (12), led to the work described in this paper. The assay we employed for protease activities, the hydrolysis of amino acyl-NAs, is conventionally used for aminopeptidases The extensive digestion of Cab proteins in membranes from pg-113 cells by the purified protease (Fig. 5) suggests that the enzyme also has endoprotease activity. This finding in vitro is consistent with the conclusion that this enzyme plays a role in accumulation of Cab proteins in vivo. However, digestion of several other proteins in pg-113 membranes indicated that the activity of the protease is not limited to breakdown of Cab proteins and may provide general proteolytic function in membranes. Its activity with the set of artificial substrates we tested suggested that the enzyme has a high affinity for nonpolar amino acid residues. A

chloroplast development. Digestion of Cab Proteins in Membranes from pg-1 13 Cells To determine whether the purified protease is capable of digesting Cab proteins, the enzyme from y-1 cells was added to thylakoid membranes prepared from pg-113 cells. As shown in Figure 5, the Cab proteins were degraded in these membranes over the time of incubation. Similar results were obtained in other experiments in which newly synthesized Cab proteins were labeled in vivo with ["4C]arginine, separated by SDS-PAGE, and degradation monitored by autoradiography. In control samples of membranes from y-1 cells, no degradation of the Cab proteins was detected during incubation with the purified protease (not shown). It is also apparent in Figure 5 that proteolytic action was not limited to the Cab proteins.

300

OH E-Et

aI)

.0_

200

C0

a

-

QL) r-

A9

lc\P0tIPrOH MeOH \o

-

100' PrOH 0

I

0

10

20

Percent Alcohol (vol/vol) Figure 6. Effects of alcohols on protease activity in membranes solubilized with 1% Triton X-100. Protease was assayed with 1.4 mM Leu-NA in 40 mm Tricine-NaOH, pH 8.0. MeOH, methanol; EtOH, ethanol; IPrOH, isopropanol; PrOH, propanol.

MEMBRANE PROTEASE FROM CHLAMYDOMONAS protease with this specificity would be well suited to digest hydrophobic regions in proteins, which normally reside in a membrane, when integration into the membrane is impaired. The stimulation of activity by alcohols (Fig. 6) is consistent with function within a hydrophobic environment. The cellular location of this protease has not been determined, although the availability of the purified protein will permit immunocytochemical localization. The insensitivity of the Chlamydomonas protease to standard inhibitors precludes its inclusion in one of the major classes of proteases. A protease in thylakoid membranes of pea was identified that cleaves precursors of plastocyanin (9, 16), which also was insensitive to typical inhibitors. However, this enzyme has not been characterized sufficiently to allow comparison with the Chlamydomonas protease. Attempts to minimize protein degradation by adding protease inhibitors to membrane preparations obviously will not control the activities of these enzymes. Other membrane-bound proteases that were identified in plant cells (5, 8, 20) have properties quite distinct from the Chlamydomonas enzyme. Also, no similarities have been found with soluble proteases identified in plant cells or chloroplasts (19, 20, 25). Inactivation of the protease by photodynamic conditions and DEPC suggested that an easily oxidized amino acid, such as a histidyl moiety, may be involved in catalysis. The pH profile (Fig. 2) also supports the involvement of at least one histidyl residue in catalysis. A histidyl residue may activate a water molecule to achieve hydrolysis of the substrate. Preliminary experiments showed a solvent deuterium isotope effect on the reaction rate of about 3, which, coupled with the pH profile, suggests general base-catalysis as the reaction mechanism (our unpublished data). Elucidation of the catalytic mechanism of the Chlamydomonas protease will be important in further defining the function of the enzyme and developing inhibitors.

10.

11. 12.

13.

14.

15. 16.

17. 18.

19. 20.

21.

22.

LITERATURE CITED

1. 2. 3. 4. 5. 6.

7. 8.

9.

Arnon D (1949) Copper enzymes in isolated chloroplasts. Polyphenol oxidases in Beta vulgaris. Plant Physiol 24: 1-15 Bassi R, Rigoni F, Giacometti GM (1990) Chlorophyll binding proteins with antenna function in higher plants and green algae. Photochem Photobiol 52: 1187-1206 Bennett J (1983) Regulation of photosynthesis by reversible phosphorylation of the light-harvesting chlorophyll a/b protein. Biochem J 212: 1-13 Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254 de Barros EG, Larkins BA (1990) Purification and characterization of zein-degrading proteases from endosperm of germinating maize seeds. Plant Physiol 94: 297-303 Dixon M, Webb EC (1979) Enzymes, Ed 3. Academic Press, New York Eichenberger W, Boschetti A, Michel HP (1986) Lipid and pigment composition of a chlorophyll b-deficient mutant of Chlamydomonas reinhardtii. Physiol Plant 66: 589-594 Girard V, MacLachlan G (1987) Modulation of pea membrane f,-glucan synthase activity by calcium, polycation, endogenous protease, and protease inhibitor. Plant Physiol 85: 131-136 Halpin C, Elderfield PD, James HE, Zimmerman R, Dunbar

23.

24.

25.

26. 27. 28.

29.

937

B, Robinson C (1989) The reaction specificities of the thylakoidal processing peptidase and Escherichia coli leader peptidase are identical. EMBO J 8: 3917-3921 Hoober JK (1987) The molecular basis of chloroplast development. In MD Hatch, NK Boardman, eds, The Biochemistry of Plants, Vol 10. Academic Press, Orlando, FL, pp 1-74 Hoober JK, Franzi J (1980) Analysis of the mechanism of photodynamic induction of synthesis of a polypeptide in Arthrobacter sp. Photochem Photobiol 32: 643-652 Hoober JK, Maloney MA, Asbury LR, Marks DB (1990) Accumulation of chlorophyll a/b-binding polypeptides in Chlamydomonas reinhardtii y-1 in the light or dark at 380C. Plant Physiol 92: 419-426 Hoober JK, Marks DB, Keller BJ, Margulies MM (1982) Regulation of accumulation of the major thylakoid polypeptides in Chlamydomonas reinhardtii at 250C and 380C. J Cell Biol 95: 552-558 Hoober JK, Millington RH, D'Angelo LP (1980) Structural similarities between the major polypeptides of thylakoid membranes from Chlamydomonas reinhardtii. Arch Biochem Biophys 202: 221-234 James GT (1978) Inactivation of the protease inhibitor phenylmethylsulfonyl fluoride in buffers. Anal Biochem 86: 574-579 Kirwin PM, Eldersfield PM, Robinson C (1987) Transport of proteins into chloroplasts. Partial purification of a thylakoidal processing peptidase involved in plastocyanin biogenesis. J Biol Chem 262: 16386-16390 Lamberts JJM, Neckers DC (1985) Rose bengal derivatives as singlet oxygen sensitizers. Tetrahedron 41: 2183-2190 Lang WC, Blatt D, Plapp R (1979) Proteolytic enzymes in Chlamydomonas I. A survey of the aminopeptidase pattern in asynchronous vegetative cells of Chlamydomonas reinhardtii. Plant Cell Physiol 20: 657-665 Liu X-Q, Jagendorf AT (1986) Neutral peptidases in the stroma of pea chloroplasts. Plant Physiol 81: 603-608 Liu X-Q, Jagendorf AT (1986) A variety of chloroplast-located proteases. In G Akoyunoglou, H Senger, eds, Regulation of Chloroplast Differentiation. Alan R. Liss, New York, pp 597-606 Maloney MA, Hoober JK, Marks DB (1989) Kinetics of chlorophyll accumulation and formation of chlorophyll-protein complexes during greening of Chlamydomonas reinhardtii y-1 at 380C. Plant Physiol 91: 1100-1106 Marks DB, Keller BJ, Hoober JK (1985) In vitro processing of precursors of thylakoid membrane proteins of Chlamydomonas reinhardtii y-1. Plant Physiol 79: 108-113. Michel H, Tellenbach M, Boschetti A (1983) A chlorophyll bless mutant of Chlamydomonas reinhardtii lacking in the lightharvesting chlorophyll a/b-protein complex but not in its apoproteins. Biochim Biophys Acta 725: 417-424 Miles EW (1977) Modification of histidyl residues in proteins by diethylpyrocarbonate. Methods Enzymol 41: 431-442 Moriyasu Y, Sakano K, Tazawa M (1987) Vacuolar/extravacuolar distribution of aminopeptidases in giant alga Chara australis and partial purification of one such enzyme. Plant Physiol 84: 720-725 Peter GF, Thornber JP (1991) Biochemical composition and organization of higher plant photosystem II light-harvesting pigment-proteins. J Biol Chem 266: 16745-16754 Sarath G, de la Mofte RS, Wagner FW (1989) Protease assay methods. In RJ Beynon, JS Bond, eds, Proteolytic Enzymes, A Practical Approach. IRL Press, Oxford, pp 25-55 Terao T, Katoh S (1989) Synthesis and breakdown of the apoproteins of light-harvesting chlorophyll a/b proteins in chlorophyll b-deficient mutants of rice. Plant Cell Physiol 30: 571-580 Tuppy H, Wiesbauer U, Wintersberger E (1962) Aminosaurep-nitroanilide als substrate fur aminopeptidasen und andere proteolytische fermente. Hoppe-Seylers Z Physiol Chem 329: 278-288