Characterization of three new chlorophyll-protein complexes

Vol. 81, No. 4, 1978 April 28,1978

8lOCHEMlCAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1113-1118

CHARACTERIZATION OF THREE NEW CHLOROPHYLL-PROTEIN COMPLEXES Fernando Henriques*and

Roderic

B. Park

Department of Botany University of California Berkeley, California 94720 Received

March

6,1978

SUMMARY Three new chlorophyll-proteins with electrophoretic mobilities intermediate between those of the P700 chl a-protein and the light-harvesting chl a,b-protein complexes are reported and their absorption spectra and polypeptide composition and II , conare characterized. Two of these chlorophyll-proteins, bands I tain approximately equal amounts of chl a and b, have polypepti 's e compositions similar to that of the light-harvesting chl a,b-protein and probably represent oligomers of the latter ccmplex. The third new chlorophyll-protein contains only chl a and its major polypeptide(s) is in the 42 kd region. Indirect evidence indicates this chlorophyll-protein is associated with the reaction-center of photosystem II. INTRODUCTION Solubilisation onic detergent gels yields

SDS followed

from wild-type

by electrophoretic

two chl-protein

light-harvesting teins

of chloroplasts

complexes:

chl a,b-protein

of these co-workers

and Hayden and Hopkins Elsewhere

(6) we reported

*Fernando

Henriques

Reports

the ani-

on SDS-polyacrylamide (CPI)

of additional

their

characterization.

Recently,

a dimer of the LHCP complex in tobacco (5) identified three

with

and the chl-pro-

(2, 3), but the low amounts and unstable

complexes have prevented (4) described

separation

plants

the P700 chl a-protein

(LHCPC) (1).

have appeared sporadically

higher

a new chl a-protein

new chl-proteins

was supported

by Biomedical

ABBREVIATIONS:CPI - P700 chl a-protein LHCPC - light-harvesting

R&my and

chloroplasts

complex in maize.

in chloroplast Research

nature

membranes of

Grant lSO7 RR 07006.

complex chl a,b-protein

complex

kd - kilodalton SDS - sodium dodecyl

sulfate 0006-291X/78/0814-1113$01,00/0 1113

Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 8 1, No. 4, 1978

romaine lettuce,

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNlCATlONS

with electrophoretic

mobilities

intermediate between those of

the CPI and LHCPcomplexes. We describe here the absorption spectra and polypeptide composition of these chl-proteins ships with other chl-proteins systems. We also describe of the new chl-proteins

previously

and discuss their

tentative

relation-

known and with the two photochemical

a procedure which reproducibly

yields large amounts

and discuss someof the factors which have probably

hindered their previous detection. MATERIALANDMETHODS Chloroplasts were isolated from lettuce (Lactuca sativa, L. var. romana, Hart.) as described before (7) and washed twic=lDTA (pH 8.6). The pellet from the last washing was homogenized in a few mls of 0.0625 M Tris-HCl, pH 6.8-5X B-mercaptoethanol-10% glycerol, the chlorophyll content was measured (8) and the suspension diluted with the samemediumto a concentration of ca. 1.5 mg chl/ml. SDSfrom a stock solution (10X, w/v) was added to a final weight ratio of SDS/chl=5 and the membraneswere solubilized in a Kontes glass homogenizer. The extent of solubilization was controlled by varying the number of passes between the pestle and wall of the homogenizer. All glassware and solutions, with the exception of the SDSstock, were ice-cold before uge. Electrophoresis was carried out immediately after solubilization, at 4 C, in the dark, for 2.5 hours. A 5% (w/v) polyacrylamide stacking gel and a 9% (w/v) separating gel were used as previously described (7). After electrophoresis, the gels were scanned at 650 or 675 nm to detect the chlorophyll-containing bands and these were cut out for further studies. Absorption spectra of chlorophyll-proteins were measured in situ in the gel slices with a Cary model 14 recording spectrophotometer. Fluorescence spectra were performed on a PerkinElmer MPF-2A spectrofluorometer equipped with a low-temperature cell. Polypeptide analysis of chlorophyll-proteins was.performed by overnight extraction of the finely dispersed gels in a minimal volume of Laemmli's buffer (9) and re-electrophoresis. RESULTS Figure 1 is a densitometric of lettuce

chloroplast

membranesafter

CPI, LHCPCand FP represent, harvesting

chl a,b-protein

three additional

tracing

of the chlorophyll-containing electrophoretic

respectively,

the P700 chl a-protein,

and the free pigment (1).

chl-proteins

observed in this work.

qualitative

pattern

racea, L.,

Nicotiana spp., Vigna unguiculta

reported here for lettuce

Phaseolus spp., Hordeumvulgare,

L. (wild-type

other species.

1114

separation.

Bands IIb,

bands Bands marked the light-

IIa and A are

We have also seen this

in chloroplasts

from Spinacia ole-

(L.) Walp, Medicago sativa,

L.,

and a chl b-less mutant) and

Vol. 8 1, No. 4, 1978

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

CPI

I I

0-I A L.HCPC

LHCPC

$1 /Jfl

CP I

4

j!( 2

640

675

710nm

Figure 1.

Densitometric tracing at 670 nm of chlorophyll-containing bands from romaine lettuce. Arrow indicates direction of eleetrophoretic migration.

Figure 2.

Room-temperature absorption spectra of chl-proteins lettuce.

from romaine

Figure 2 presents the room-temperature absorption spectra of the five major chl-proteins

of Figure 1.

The spectra for CPI and LHCPCare identical

to

those previously

reported for these complexes (1) with a single maximumat 676

nm for CPI and maxima at 672 nm and 652 nm for LHCPC. Like LHCPC,bands IIb and IIa display maxima at 672 and 652 nm. There is a small, but reproducible difference tion.

amongthese three complexes in the ratio

This ratio

is lowest in peak IIb,

of the 672:652 nm absorp-

higher in peak IIa and highest in LHCPC.

As the shoulder at 652 nm is due to chl b, the increasing ratios increase in the amount of chl a relative

indicate

an

to chl b in progressing from bands IIb

to IIa to LHCPC. The chl-protein

of band A, which accounts for about 5% of the total

shows only one maximumat 671 nm indicating riched in chl a.

that this complex is greatly

Comparedwith CPI, the band A peak is shifted

chl, en-

about 5 nm

towards short wavelengths and this complex does not possess the low temperature

1115

Vol. 81, No. 4, 1978

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ji :: i; jI ii :.‘: i’ 1: i ;

;i: I0.1

Figure

3.

Low-temperature fluorescence light:410 and A. Excitation

Figure

4.

Densitometric tracing at 560 nm of polypeptides IIa (---) and IIb (**a).

long-wavelength Figure

fluorescence

emission

4 shows the polypeptide

and A; the LHCPC and CPI profiles

emission nm.

of CPI (Figure composition

Band IIb similarly

few other

peptides

displays

other

of chl-proteins

3).

before

peptides

60-70 kd peaks in band IIb may partly

II,,

IIb

Band IIa 25 and 23

together

with

a

The 53 kd peak in band IIa and the

represent

Ill6

(7).

at 27.5,

the 25 and 23 kd components,

in the 60-70 kd region.

CPI

from bands A (-),

of the chl-proteins

have been discussed

shows a major component at 53 kd and three kd.

spectra

A

undissociated

complexes but

Vol. 81, No. 4, 1978

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

mostly

include

plexes

and extracted

profile

peptides

together

of band A contains

shoulder. this

extraneous

with

identical

to the original

these from the gel slices.

a major 42 kd component with

The minor peptide

profile

of mobilities

The polypeptide

a lower

molecular

peaks at 25 and 23 kd are probably

by the tailing

com-

weight

contributed

to

of LHCPC.

DISCUSSION Of the three

new chlorophyll-proteins

are shown to be related latter

complex.

respectively,

must remain conjectural exist

The third

to the LHCPC and probably

A simple interpretation

dimer and trimer,

complexes

we know whether

composition

but some indirect

center

of Anthirrhinum

majus defective

of photosystem

Chua and Bennoun (11) similarly

subchloroplast fraction

of Klein

control

enrichment

chloroplasts

band A and, thus, tion

center

II.

making up the three

spectrum

known chlorophyll-protein.

indicates

in the it may be related

Herrmann (10) showed that

weight

II activity

lacked

of about 45 kd.

in photosystem

lacking

a mutant

a polypeptide

In Chlamydomonas, or with

II activity,

or the F-11 fraction of peptides

to be correlated

decreased

of Wessels

and Borchert

in the 45 kd region,

as compared

This polypeptide(s) with

of photosystem

indirectly

photosystem

associate

Also

such as the TSFII-a

of about

II activity

to the 42 kd component of our chlorophyll-protein

these observations

The visualization

evidence

or PSI preparations.

ident,ical

however,

or had reduced amounts of a 47 kd peptide.

enriched

45 kd found by these workers most probably,

of the

IIa and IIb are a

an absorption

showed that mutants

and Vernon (12),

(13) show a substantial with

lacked

fractions

oligomers

new chlorophyll-protein

in photosystem

to have a molecular

II activity

the peptides

from any other

played by this

to the reaction

photosystem

IIb and II,,

This interpretation,

band A, possesses

distinct

We do not yet know the role

which we estimate

complexes

two,

the same ratio.

chlorophyll-protein,

process,

here,

represent

is that

of the LHCPC.

until

in exactly

and polypeptide

photosynthetic

characterized

band A with

is, of

the reac-

II.

of additional

chlorophyll-proteins

1117

in this

work

is pro-

Vol. 8 1, No. 4, 1978

BIOCHEMICAL

AND BIOPHYSICAL

bably due to the more gentle solubilization immediate electrophoretic tures.

The traditional

RESEARCH COMMUNICATIONS

of chloroplast

separation of solubilized

membranesand the

material,

at low tempera-

procedure undoubtedly leads not only to solubilization

of membranesubunits into their

smallest constituents associations.

but also to dissociation

of more labile

chlorophyll-protein

the additional

complexes seen here, but also from the relatively

of free pigment (520%) seen in our experiments.

This is evident not only from

In addition,

small amounts our solubiliza-

tion procedure, leaving part of the LHCPcomplex in its oligomeric form, reduces the amount of the LHCPCmonomerwhich in large amounts tends to tail obscure band A.

Finally,

the short electrophoretic

workers (1) do not allow spatial

distances used by other

separation of band A from neighboring LHCPC.

These data support the view that chlorophyll-protein membranesare large complex structures ditionally

that further

associations in intact

compared with the minimal subunits tra-

isolated by the SDSsolubilization

We anticipate

and

and electrophoretic

experiments with milder isolation

help bridge the gap between chlorophyll-proteins

techniques.

techniques will

and the intact

photosynthetic

membrane. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

Thornber, .I. P. (1975) Ann. Rev. Plant Physiol. 26, 127-158. Herrmann, F. and Meister, A. (1975) Photosynthetica 6, 177-182. Hiller, R. G., Genge, S. and Pilger, D. (1974) Plant Science Letters 2, 239-242. RGmy,R., Hoarau, J. and Leclerc, J. C. (1977) Photochem. Photobiol. 26, 151-158. Hayden, D. B. and Hopkins, W. G. (1977) Can. J. Bot. 55, 2525-2529. Henriques, F. and Park, R. B. (1978) Arch. Biochem. Biophys. (in press). Henriques, F. and Park, R. B. (1977) Plant Physiol. 60, 64-68. Arnon, D. I. (1949) Plant Physiol. 24, l-15: Laemmli, U. K. (1970) Nature 227, 680-685. Herrmann, F. (1972) Exptl. Cell Res. 70, 452-453. Chua, N. H. and Bennoun, P. (1975) Proc. Nat. Acad. Sci. U.S.A. 72, 21752179. Klein, S. and Vernon, L. P. (1974) Photochem. Photobiol. 19, 43-49. Wessels, J. S. and Borchert, M. T. (1974) Proc. Third Int. Cong. Photosynt. pp. 473-484- Elsevier Scientific Publishing Company, Amsterdam, The Netherlands.

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