The structure of a «simple phycobilisome

Ann. Microbiol. (Inst. Pasteur) 1983, 134 B, 159-180

THE OF

A

STRUCTURE

(( S I M P L E

)) P H Y C O B I L I S O M E

by A. N. Glazer (~), D. J. LundeU (~), G. Y a m a n a k a Q) (*) and R. C. Williams (2)

(~) Department o/Microbiology and Immunology, and (~) Department o/Molecular Biology, University o/Cali[ornia, Berkeley, Cali[ornia 94720 ( U S A )

SUMMARY This report describes the properties of a relatively simple phycobilisome,

Synechococcus 6301 (Anacyslis nidulans). Morphology. - - Examination of wild t y p e and m u t a n t phycobilisomes by electron microscopy has shown them to have two morphologically differing substructures when seen in ,r face-view ~. There is a core consisting of two contiguous objects, disc-like in face-view projection, 115 ~_ in diameter, and six rods, each composed of several stacked discs 60 ~ thick and 120 .~ in diameter, which radiate from the core in a hemidiscoidal arrangement. Each of the core components consists of four discs ~ 30 ~ thick. Rod substructures. - - Each of the discs in the rod substructure is a phycocyanin hexamer held together by interaction with a specific linker polypeptide, i. e., it has the composition ( ~ ) ~ . X , where X is the linker polypeptide and ~ a phycoeyanin monomer. The disc proximal to the core is an (~)6" 27,000 complex. A small portion, Mr ~ 2,000, of the Mr 27,000 polypeptide is essential to the attachment of this disc to the core. From studies of phycobilisomes from nitrogen-starved cells, and from m u t a n t s containing lowered amounts of phycoeyanin relative to allophycocyanin, the second disc has been established to be an (~)s.33,000 complex. Either (~)6" 33,000 or (~)6" 30,000 complexes occupy the positions in the rods distal to the (~)8" 33,000 discs. Core substructure. - - Structural studies on the core and on core-rod junctions were greatly facilitated by the isolation of a mutant, strain A N l l 2 , which produces phycobilisomes with rods only one disc in length but with normal cores. Partial dissociation of these incomplete phycobilisomes under a variety of conditions, and separation and characterization of the resulting sub-complexes, has led to the determination of the composition of four distinct ~ trimeric ~ complexes, each of which is present in two copies per phycobitisome. These complexes, which account for the composition of the core, are as follows: ( ~ ) ~ . 10,500 with ~'max F at F ~ at 660 nm; /'(xAP~ 662 nm; (~)AP with kma ~ ~ ApB~AP~ ea z" 10 ,500 with k.... at 680 nm; Manuscrit recu le 23 mars 1983. (*) Present address: Department of Structural Biology, Stanford University School of Medicine, Stanford, California 94305 (USA).

160

A. N. GLAZER AND COLL.

where ~*P and , P B are ~ subunits of allophycocyanin and allophycocyanin B, respectively, and ~*v is a subunit common to these two biliproteins; F (~)~P.18,300.40,000" 9 ll,000* with X..... at 680 rim, where the Mr 40,000* and ll,000* polypeptides are derived from a Mr 75,000 polypeptide by tryptic digestion. Structure-/unction relationships. - - Interaction of phycocyaniu with the linker polypeptides leads to a red shift in the spectrum of the biliprotein with an increase in long-wavelength absorbance and an increase in the fluorescence quantum yield9 The fluorescence emission maxima of the phycocyanin complexes with the Mr 30,000, 33,000, and 27,000 polypeptides lie at 643, 648, and 652 nm, respectively 9 Thus, energy migration within the rod substructure is preferentially directed towards the core. The pathway of energy transfer within Synechococcus 6301 phycobilisomes deduced from the above studies is

(cr 6.33,000> (~r (~r 30,000

33,000 -->(~r

AP > APB 27,000~ff ? ~ :~ Mr 18,300 --->M~ 75,000 polypeptide

polypeptide

KEY-WORDS: Phycobilisome, Ultrastructure, Assembly, Energy transfer.

INTRODUCTION.

Biliproteins contribute to the attractive and varied colors which are characteristic of the cells of cyanobacteria and the chloroplasts of red algae. In cyanobacteria, they may constitute up to 50 % of the soluble protein of the cell. In quantity, phycocyanin (Xmax ~ 620 nm) and phycoerythrin (~. . . . ~ 565 nm) are the major of these proteins9 A third, less abundant but universally present biliprotein, allophycocyanin (X.... 650 rim), was recognized only in the 1950s. A hundred years ago, Engelmann [4] showed that light absorbed by these intensely-colored proteins promoted oxygen evolution. It is now generally accepted t h a t the biliproteins make up the bulk of the antenna for photosystem II and make some contribution to the action spectrum of photosystem I [24, 29]. At this time, the precise physical interrelationship between biliproteins and photosystems II and I remains a matter for speculation9 When cyanobacterial or red algal cells are ruptured, the biliproteins are released in water-soluble form. These well-characterized chromoproteins are a family of proteins descended from a common ancestral gene [12]. The building block of each of these proteins is a monomer, ~ , containing two dissimilar polypeptide chains, each bearing covalently attached tetrapyrrole prosthetic groups9 The stable in vitro assembly forms of these proteins are the disc-shaped trimer, (~)3, and hexamer, (~)~ [10]. The high efficiency of energy transfer from the biliproteins to chlorophyll a observed in intact cells mandates a close association between these proteins and the photosynthetic lamellae. The studies of Gantt and her coworkers (see refs. [5 and 8] for reviews) demonstrated t h a t within the cell, the biliproteins form supramolecular aggregates, phycobilisomes, which are attached in regular arrays to the photosynthetic lamellae. The morphology of phycobilisomes (e. g. fig. l) as well as their biliprotein composition vary depending on the source organism [2, 5, 6, 15, 17, 25]. Gantt et al. [7, 9] developed procedures for the isolation of intact phycobilisomes. The major criteria for the intactness of the isolated particles were retention of energy transfer among the constituent biliproteins and preservation of the organization of the particles as assessed by electron microscopy. Investigation of the composition of a variety of phycobilisomes indi-

STRUCTURE OF A (( SIMPLE )) PHYCOBILISOME

161

cared that, in addition to the phycobiliproteins, they contained a number of <(uncolored )) polypeptides [28, 32] as well as previously unrecognized minor biliproteins [21, 23, 27]. In this report, we present an account of the analysis of the structure of the phycobilisome of a unicellular eyanobacterium Sgnechococcus 6301 (Anacgslis nidulans). This phycobilisome was chosen for detailed study for two principal reasons: the relative simplicity of its biliprotein composition (it contains only phycocyanin and allophycocyanin as major biliproteins); and its ultrastructure, which appeared to be simpler than t h a t of other phycobilisomes (fig. 1). The simplicity of the structure of this phycobilisome is only relative. The analysis has revealed an extraordinarily sophisticated assembly. In addition to phycocyanin and allophycocyanin, the Sgnechococcus 6301 phycobilisome contains three quantitatively minor but structurally and functionally very important biliproteins, and six uncolored polypeptides which function in the assembly of the particle. Multiple copies of these components form the particles seen in figure 1 A. U L T R A S T R U C T U R E OF T H E

Synechococcus 6301

PHYCOBILISOME.

The Syuechococcus 6301 phycobilisome exhibits two morphologically differing substructures when seen in (( face-view )). There is a core consisting of two contiguous objects, disc-like in face-view projection, 115 A in diameter, from which radiate in a hemidiscoidal array six rods, each composed of several stacked discs 60 ~ thick and 120 /~ in diameter (fig. 1 A) [15, 33]. The number of discs per rod varies from one to as many as seven in a single phycobilisome, and the average rod length is dependent on culture conditions and the nutritional state of the cells. Each of the core components consists of four discs ,~ 30 ~ thick [33]. The morphology of other phycobilisomes is more complex. Those of the majority of cyanobacteria [2, 15] and of the red alga Rhodella violacea [25] have a core made up of three cylindrical units in an equilateral array (for an example, see fig. 1 B). The details of the morphology of other red algal phycobilisomes such as those shown in figure I C and D are hard to discern because of superposition of structural elements. COMPOSITION.

Accurate determination of the polypeptide composition of the Synechococcus 6301 phycobilisome (table I) presented a challenge. The subunits of phycocyanin and allophycocyanin are present at ,~ 100 and ~ 20 copies per phycobilisome, respectively, whereas several other polypeptides are present in two copies or fewer. Moreover, the molecular weights of four of the polypeptides are similar; they fall into the range of 17,700 to 18,300. The values presented in table I are a composite of analyses of wild-type phycobilisomes [31, 32], phycobilisomes from a mutant strain AN112 which produces rod substructures only a single disc in length [33], a rod-core junction 18 S subassembly particle [20, 34] and of individual complexes derived from the core substructure [20, 21]. Application of SDS-polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing, alone and in combination, to these various structures permitted resolution of all of the components. Since all of the carbon in cyanobacteria can be derived from CO~, phycobilisomes from cells grown with 14CO2 were used for the preparation of labelled subassembly particles and polypeptide quantitation was performed by scintillation counting [20]. ROD

SUBSTRUCTURES.

The rod substructures were investigated in several different ways: a) examination of the effect of early stages of nitrogen starvation on phycobilisome strucAnn. 51ierobiol. (Inst. Pasteur), 134 B, n ~ 1, 1983.

11

162

A. N. G L A Z E R A N D

"]'ABLE I.

-

-

-

COLL.

Composition of (( Syneehoeoeeus 6301 )) phyeobilisomes. N u m b e r c f copies

Mr •

10 ~

Wild type

ANll2

Phycocyanobilins per p o l y p e p / i d e

Rod substructure 19.0 PC-~ (a) 17.7 PC-~ 33 3O 27 12

~ 100 ~ 100 ~ 6 3-6 6 n. (I.

36 36 0 0 6 0

2 1 0 0 ~) 0

20 22 2 2 2 4

20 22 2 2 2 4

1

(',ore substructure 18.2 A P - ~ 17.6 AP-O 17.7 A P I t - u 18.3 75 10.5

1 1 1 1 o

Location undetermined 45

<1

<1

0

(1) A b b r e v i a t i o n used are: PC = p h y c o c y a n i n ; A P = a l l o p h y c o c y a n i m A P B = a l l e p h y c o c y a n i n B; n. d. = n o t determired.

ture; b) analysis of phycobilisomes from mutants which make incomplete particles; c) examination of phycobilisomes from cells grown under different culture conditions; and d) in vitro assembly of the rod substructures from purified polypeptide components.

N ilrogen slarvalion. When cyanobacteria are transferred to a medium lacking in combined nitrogen, the cells degrade phycobiliproteins [1] and synthesis of phycobiliproteins ceases [18]. In the initial phase of nitrogen starvation, several simultaneous changes were seen in the phycobilisome: a) a decrease in sedimentation coefficient (fig. 2 A); b) a decrease in the phycocyanin/allophycocyanin ratio; c) a shift in the fluorescence maximum from 673 to 676 nm (fig. 2 B); and d) a selective loss of a Mr 30,000 nonpigmented polypeptide (fig. 2 C) and some 30 % of the phycocyanin [30]. It is interesting to note that under these conditions, significant differences exist in the rates of proteolytic degradation of different phycobiliproteins within the cell. Under normal culture conditions, a small amount of free phycocyanin is present in the cells and free allophycocyanin is virtually undetectable. In contrast, during early stages of nitrogen starvation, the free phycocyanin is totally degraded and a trace of free allophycocyanin is detected (fig. 2 D).

FIG. 1. - - Electron micrographs of phgcobilisomes o[ cganobaeleria and red algae.

The sources of the p h y c o b i l i s o m e s are the c y a n o b a c t e r i a Sgnechococcus 6301 (A), a n d Sgnechocgstis 6701 (B), a n d the red algae Porphgridium eruentum (C) a n d Gastroclonium coulteri. Bat" represents 0.1 v m .

FIG, 1

1~'I6. 2. - - Effect o{ nitrogen starvation on the properties of Synechococcus 6301 phgcobilisomes. A. - - Sucrose step gradient profiles after centrifugation of the soluble s u p e r n a t a n t fractions from control cells and cells which were starved two days for combined nitrogen (see [30] for experim e n t a l details). B. - - Corrected fluorescence emission spectra of phycobilisomes from control cells (n) or nitrogenstarved cells (o). C. - - P A G E in the presence of SDS of phycobiliscmes from control and nitrogen-starved (-NO3) cells. D. - - A b s o r p t i o n spectra of phycobiliprotein regions (Bands 2) of the sucrose density gradients s h o w n in p a r t A. A b s o r p t i o n m a x i m a are at 624 n m for the control (Baud 2a) and 654 n m for the phyeobiliprotein from nitrogen-starved cells. D a t a from [30].

'.......

A

1.0

a

b

i

SompteW ~

/

O.2M ~'

0.4M U

/

[Ba.d 2]

h~-~-obillsomN

)6

[B~ ,]

0.6M

//

0.SM

).4 1.0

!

B

\

M

Control

)2

2 Days of

Starvat ion for Combined Nitrogen

Oc 600

Mr x 10-3 I

D 0.2

75-45--

iil~i!:~~i{i~~:~:i!i~i i~!i i~

65O 700 W a v e l e n g t h (nm) I

I

750

1

Band 2b--~/

Band Za ~

A

/

0

:3:3_

o.1

27--

< ///I

19-17.7

j

_

500

o

C)

z

I

Fro.2

600 W a v e l e n g t h (rim)

I

700

STRUCTURE OF A (( SIMPLE )) PHYCOBILISOME

167

From the above results, it was concluded that the Mr 30,000 polypeptide is closely associated with peripheral phycoeyanin discs in the phycobilisome. Moreover, the particle remaining after the loss of these components shows unimpaired energy transfer (fig. 2 B).

Analysis o/incomplete phgcobilisomes. Two nitrosoguanidine-induced mutants were isolated from Synechococcus 6301 [31, 33]. These mutants, A N l l 2 and AN135, produced abnormally small phvcobilisomes~ containing only 35 and 50 O//oof the wild-type level of phycocyanin, respectively. AN135 phycobilisomes contain less Mr 33,000 polypeptide than wild type and the Mr 30,000 polypeptide can only be detected in phycobilisomes from cultures grown under conditions favoring high levels of phycocyanin [31]. A N l l 2 lacks both the Mr 30,000 and 33,000 polypeptides and produces phycobilisomes of constant size and composition, independent of growth conditions. In other respects, the compositions of the mutant phycobilisomes are similar to those of the wild type. The morphology of the core appears to be normal in the phycobilisomes of both mutants. However, the rod substructures of AN112 phyeobilisomes are only one disc in length, and those of AN135 do not exceed two discs (tig. 3). These observations are consistent with the view that the Mr 30,000 and 33,000 polypeptides as well as phycocyanin are associated with the rod substructures. Comparison of A N l l 2 phycobilisomes with those of AN135 indicates that the M,. 33,000 polypeptide is associated with the second phycocyanin disc distal to the core in the rod substructure.

Fro. 3. - - Electron micrographs of phgcobilisome particles from mutants which make incomplete structures. A ~ P h y c o b i l i s o m e s from m u t a n t AN135. B = P h y c o b i l i s o m e s from m u t a n t AN112 [33]. Inset shows an enlarged view of an A N l 1 2 p a r t i c l e s w i t h the core-cylinder axes in t h e plane of the m i c r o g r a p h . One of t h e core c y l i n d e r s clearly e x h i b i t s four t h i n parallel s u b s t r u c t u r e s which are p r o b a b l y discs seen on edge. B a r r e p r e s e n t s 0.1 p.m.

Influence o/ culture conditions. Among the responses of Sgnechococcus 6301 to various environmental conditions is the alteration in the level of phycocyanin per cell. High levels of phycocyanin are favored by conditions of high temperature, light intensity and COs concentration [3, 16, 26, 31]. Analysis of phycobilisomes showed that the molar ratio of

168

A.N.

GLAZER AND COLL.

phycoeyanin to allophycocyanin in wild-type phycobilisomes could be varied over about a two-fold range by alterations in culture conditions with parallel changes in the amounts of the M~ 33,000 and 30,000 polypeptides, whereas the levels of two other polypeptides, Mr 27,000 and 75,000, did not vary [31]. Besults obtained with wild-type and mutant phycobilisomes are shown in figure 4. From such data, it was possible to calculate that approximately one M~ 33,000 or 30,000 polypeptide is present per phyeoeyanin hexamer (~)6.. Wild-type cells and those of AN135 were able to produce phycobilisomes varying m phycoeyanin content. The phycocyanin A

~

I

1

I

I

d

5.0 4.0 o.

s.o 2.0

1.0 n Y2

I 9 At

0.3

I

O 27

kD

75

kD

9

02

"6 0.l o_

o

I

n

o

0"

I

~

~

0

,I

0.~ r', oa

02.

33

kD

"~e

J

02 O

0- 0. I f f I

I.E

f

1.6

2.0

2.4

2.8

A624:A654 Fro. 4. - - R e l a t i o n s h i p between relative p o l g p e p l i d e levels and the ratio of 6 2 t n m to 654 n m absorbance (A62 ~ : A6~4) in phgcobilisomes. Cells were cultured u n d e r conditions favoring either high or low levels of p h y c o c y a n i n . E a c h p o i n t represents a single phycobilisome sample, characterized b y the ratio of absorbanee at 624 to 654 n m and the level of a given polypeptide normalized w i t h respect to the allophycocyanin ~ subunit. Phycobilisomes are from Sgneehococeus 6301 (o, e), AN135 (D, I ) , A N l l 2 (/~, A), and AN139 (white and black hexagons). Mutants A N l l 2 and AN139 lack the M r 30,000 and 33,000 polypeptides. A ~ Ytelationship of the p h y e o c y a n i n to allophycocyanin ratio (obtained using values for the p h y c o c y a n i n ~ s u b u n i t ) and A+2t : An~4B = Mr 27,000 and 75,000 polypeptides vs. A624 : A~54. C = Mr 33,000 and 30,000 polypeptides vs. A62~ : A+s4. A b b r e v i a t i o n s used are: kD = kilodaltons; A P - allophycocyanin; PC = p h y e o c y a n i n . D a t a from [31].

STRUCTURE OF A (( SIMPLE )) PHYCOBILISOME

169

content of A N l l 2 phycobilisomes was invariant even though an increase in the amount of free phycocyanin per cell was noted under conditions that favored formation of high levels of phycocyanin [31]. These results show that the M~ 33,000 and 30,000 polypeptides are required for extension of the rod substructures beyond the basal disc. In vitro assembly of rod substructures. Procedures were developed for purification of the M~ 75,000, 33,000, 30,000, and 27,000 polypeptides [22, 23]. These involved fractionation of phycobilisome polypeptides in the presence of denaturing agents. The purified polypeptides were insoluble in aqueous buffer solutions, but could be readily dissolved in concentrated urea or guanidine. In contrast to the acidic biliproteins, the M~ 27,000, 30,000 and 33,000 polypeptides were basic and each yielded a distinctive tryptic peptide map and amino acid composition [22]. Assembly experiments were performed by mixing these polypeptides, singly or in various combinations, with phycocyanin in acid urea. This was followed by a series of dialysis steps terminating with dialysis against 0.6 M NaK-phosphate, pH 8.0, a buffer in which Synechococcus 6301 phycobilisomes are stable. Under these conditions, the Mr 27,000, 30,000 and 33,000 polypeptides assembled phycocyanin into ordered aggregates, whereas the M,. 75,000 polypeptide did not interact with phycocyanin. In the presence of the M,. 33,000 and 30,000 polypeptides, phycocyanin assembled into hexameric discs and rods of stacked discs with the ultrastructural characteristics of the rod substructures of intact phycobilisomes (fig. 5 B and 6). Phycocyanin alone did not form such structures under the conditions used. Interaction of phycocyanin with the Mr 27,000 polypeptide led solely to the formation of hexameric discs which did not assemble into rods (fig. 5 A). Moreover, rods formed by interaction of phycocyanin with the M~ 30,000 and 33,000 polypeptides in the presence of the M~ 27,000 polypeptide were much shorter than those formed in its absence (fig. 5 C). This suggested that addition to growing rods of discs formed from phycocyanin and the M, 27,000 polypeptide terminates rod assembly. Consonant with their role

FIG. 5. - - Electron micrographs of complexes between phgeocganin and linker polgpeptides. A -- phycocyanin plus Mr 27,000 polypeptides; B = p h y c o c y a n i n plus M r 30,000 and 33,000 polypeptides; C = phycoeyanin plus M r 30,000, 33,000 and 27,000 polypeptides. All complexes were in 0.6 M N a K - p h o s p h a t e , p H 8.0. Bar represents 0.1 ~zm. For experimental details, see [22].

170

A.N.

GLAZER AND COLL.

Fro. 6. - - Electron micrograph o/rod complexes lormed by the interaction of phycocyanin with the 31 r 33,000 polypeplide. Complexes were formed in 0.6 M N a K - p h o s p h a t e , p H 8.0. N o t e t h a t t h e rods f r e q u e n t l y t e r m i n a t e in a half-disc. Bar r e p r e s e n t s 0.1 ixm.

in the assembly of phycocyanin, the Mr 27,000, 30,000 and 33,000 polypeptides were named (( linker polypeptides )) [22]. Analysis of the ordered structures formed upon interaction of phycocyanin with individual linker polypeptides showed the presence of at least one equivalent of linker polypeptide per (~)~ hexamer of phycocyanin [22]. In vitro assembly experiments, in conjunction with the studies of incomplete phycobilisomes, indicated that the (a~)e'27,000 disc interacts with the phycobilisome core and that the rod substructures are built up by successive addition of (a~)6"33,000 and (a~)~.30,000 complexes to this disc. These inferences were strengthened and the roles of the linker polypeptides in the assembly process defined more precisely in parallel studies on the rod substructures of Anabaena variabilis phycobilisomes described elsewhere [11, 35, 36]. CORE SUBSTRUCTURE AND THE CORE-ROD JUNCTION.

Isolation of mutant ANll2 phycobilisomes in which only the basal disc of the rods is present greatly facilitated determination of these portions of the structure. In addition to the subunits of allophycocyanin and phycocyanin, this phycobilisome contains the three minor biliproteins: the Mr 18,300 polypeptide plus the terminal energy acceptors (see below), the allophycocyanin B a subunit [19] and the Mr 75,000 polypeptide [23] (table I and fig. 7 A). Sucrose density gradient centrifugation of ANll2 phycobilisomes partially dissociated in 50 mM N-[Tris(hydroxymethyl)methyl]glycine,5 mM CaCI~, 10 %

STRUCTURE

OF A (( SIMPLE ,) P H Y C O B I L I S O M E

171

( w / v ) glycerol, pH 7.8, separated three fractions sedimenting at 18 S, 11 S, and

--~ 6 S, respectively. Two-dimensional maps of these fractions are shown in figure 7 B-D. Their compositions have distinctive features. The Mr 18,300 polypeptide was present only in the 18 S fraction, whereas the allophycocyanin B subunit was present in the 6 S fraction with only a trace in the 11 S fraction. The Mr 27,000 polypeptide was present in the 11 S and 18 S fractions but absent from the 6 S fraction, and M,, 75,000 polypeptide was present only in the 18 S fraction. IEF oo O 09

--

A

B

C

13

9

l

o

Fro. 7. - - Two-dimensional mapping of A N l 1 2 phgcobilisomes and of fractions obtained from these phgcobilisomes by partial dissociation. A N l l 2 phycobilisomes were partially dissociated in 50 mM tricine, 5 mM CaC12, 10 % (w/v) glycerol, p H 7.8. The resulting m i x t u r e of components was resolved into three fractions of 6 S, 11 S and 18 S by sucrose density gradient centrifugation. Polypeptide maps are as follows: A = AN112 phycobilisomes; B = ~ 6 S fraction; C = 11 S fraction; D = 18 S fraction. N u m b e r s in A represent phycocyanin ~ s u b u n i t (1), phycocyanin ~ subunit (2), allophycocyanin a s u b u n i t (3), allophycocyanin [3 s u b u n i t (4), allophycocyanin B ~ subunit (5), u n k n o w n c o m p o n e n t which m a y be derived from a p h y c o c y a n i n subunit (6) and Mr 18,300 polypeptide (7). The open circles in B and C mark the position of the absent spot 7, and the open circle in D marks the position of the absent allophycocyanin B u subunit (spot 5). I E F designates the isoelectric focusing dimension and SDS the SDS-polyaerylamide gel electrophoresis dimension. The Mr 27,000, 75,000 and 10,500 polypeptides do not enter this gel because of their basic isoelectric points. Data from [34].

The 18 S subassembly. In the analytical ultracentrifuge, the 18 S fraction sedimented as a single boundary with no evidence of heterogeneity. The fluorescence emission spectrum of this particle showed a near-identity to t h a t of intact A N l l 2 phycobilisomes, with components at 680 and 660 nm [34]. The composition of the 18 S

172

A. N. GLAZER AND COLL. TABLE II.

Composition of the 18 S subassembly by analysis

of uniformly [~4C]-labelled particles [20] (~).

Polypeptkle Allophycocyanin [3 s u b u n i t Allophycocyanin a s u b u n i t -I- M r 18,300 Phycocyanin u s u b u n i t Phycoeyanin [~ s u b u n i t Mr 27,000 Mr 75,000

Number per 18 S particle (2) 4.9 6.2 6.1 5.9 2.0 0.94

• ~ i •

0.4 0.2 0.3 0.1

= 0.2

(~) Values normalized to 2 copies of the Mr 27,000 polypeptide per 18 S particle. (2) Average of 14 determinations.

particle (table II) [20, 34] led to a molecular weight of ---550,000, compatible with the observed sedimentation coefficient. From this molecular weight and the observation t h a t the 18 S fraction contains ,--50 % of the total allophycocyanin of the A N l l 2 phycobilisome, it is evident t h a t two copies of this subassembly are released from each phycobilisome [34]. It will be noted that the ratio of the Mr 27,000 polypeptide to phycoeyanin monomer ( ~ ) in tile 18 S particle is 1/3 (table II) as contrasted to 1/6 for the A N l l 2 phycobilisome (table I). This ratio and the spectroscopic properties of the 18 S particle are compatible with the conclusion t h a t the phycoeyanin in this particle is present as two (~)3"27,000 complexes [34]. The fact t h a t native Syneehoeoeeus 6301 allophyeoeyanin and phyeocyanin are resistant to trypsin [22] was exploited by using limited tryptie digestion to produce subeomplexes of the 18 S particle. Chromatography of the products resulting from limited trypsin treatment of the 18 S particle led to the isolation of three subcomplexes: a mixture of (~)3.22,000 and (a~)3.24,000 phycocyanin complexes, an (~)3 altophycoeyanin trimer, and an allophyeoeyanm (~)~.18,300. (40,000 911,000) complex [20]. The Mr 22,000 and 24,000 polypeptides were products of degradatien of the Mr 27,000 polypeptide, whereas the Mr 40,000 and 11,000 components were fief• from the Mr 75,000 polypeptide [20].. The Mr 75,000 polypeptide had earlier been snown ~o be a bihprotein with an emission peak at 676 nm, carrying one phycoeyanobilin [23]. The phyeoeyanobilin was located on the M~ 40,000 tryptie fragment [20, 23]. The fluorescence emission maximum of the (~)2.18,300.(40,000.11,000) complex was located at 680 nm I20]. Structures deduced for the two allophycocyanin-eontaining complexes 1 and 2 isolated from the 18 S particle as well as t h a t for the 18 S particle itself are illustrated in figure 8 C and D.

The Mr 18,300 polgpeptide The Mr 18,300 polypeptide is similar in molecular weight and isoeleetrie point to other biliprotein subunits and was only detected as a phycobilisome component on two-dimensional gels (fig. 7) [34]. On one-dimensional SDS-polyacrylamide gels, it was not adequately resolved from the subunits of the more abundant biliproteins. It carries a single, covalently-bound phycocyanobilin [20]. Determination of the amino-terminal sequence of the Mr 18,300 polypeptide showed it to be homologous to the ~ subunit of allophycocyanin [20]. However, from the aminoterminal sequence and tryptic peptide maps, it is evident t h a t this is a previously undescribed bilipro.'ein. The absorption spectrum of the isolated Mr 18,300 poly-

STRUCTURE

OF A cc SIMPLE , PHYCOBILISOME

A.

Strain AN 112 Phycobilisome

C.

18S Particle

173

B,

43

2:t

Bottom View

(13AP'~,AP

a~P,B:P-18.:5K (40K']IK)

aApSaAp

I

AP

2 '83'i0"5K

AP

aP

a 3 ,B5 . 10.5K

Pc Pc

a 3 ,8:5 ' 27K

Xmax

nm

650

665

652.5

652.5

640

Ernax

mM-t crr -I

770

II00

820

1020

1000

kFox

nm

660

680

680

662

654

FIG.

8. --

A

schematic diagram o[ the A N l l 2 phycobilisome.

A pair ol a l l o p h y c o c y a n i n - e o n t a i n i n g core ,( cylinders ,, is surrounded by a hemidiscoidal array of six p h y c o e y a u i n - c o n t a i n i n g discs (~PC~PC)~ 27,000. The h e a v y lines in A indicate the portions of the structure believed to constitute the 18S particles. B shows an isometric v i e w of the core cylinders. A schematic diagram of the 18S particle is s h o w n in C. The e o m p o s i t i o n and spectroscopic properties of the subcomplexes t h a t m a k e up the 18S particle and the core cylinders are presented in D. Abbreviations are: AP = allophycocyanin; AP-B = allophyeoeyanin B; PC = phycocyanin; 18.3K, 10.5K, etc., polypeptides of Mr 18,300, 10,500, etc.

peptide is similar to that of the allophycocyanin ~ subunit. Its role is not clear, but a speculative suggestion is that it mediates the assembly of allophycocyanin monomers with one of the domains of the Mr 75,000 polypeptide in the formation of the unusual a~w~p. 18,300.75,000 complex, from which complex 2 of figure 8 is derNed. The ~ 6 S and 11 S fractions.

Chromatographic fractionation of the components of the ~ 6 S fraction led to the purification of two allophycocyanin-containing complexes (see fig. 8 D, 3 and 4) [21]: (0~AP~AP)~'10,500 a n d (~1 " APu ~ APnAP, p~ ).10,500, where QcAP and ~AP represent the ~ and ~ subunits of allophycocyanin and m'U the a subunit of allophycocyanin B. The spectroscopic properties of these complexes are given in figure 8. The M,. 10,500 polypeptide in the two complexes appears to be the same as indicated by the identity of tryptic peptide maps of this polypeptide isolated from each of the two complexes [21]. T h e complex (~1APB e~AP ~;Ap ).10,500 deserves special eomment. A protein named

174

A.N.

GLAZER AND COLL.

allophycocyanin B, with a fluorescence emission maximum at 680 nm, was originally isolated upon extensive purification of the biliproteins of Sguechococcus 6301 [13]. Subsequently, we have demonstrated t h a t allophycocyanin B, (~APR~AP)3 and allophycocyanin (~AI'~AI')3 share a common ~ subunit and t h a t the distinctive spectroscopic properties of the two biliproteins depend solely on the unique ~ subunits [19]. Moreover, rapid subunit exchange in mixtures of ( ai,~Ap)~ and (~APB~AP)a led to the formation of a statistical distribution of heterologous trimers. Calculation showed that a single a r B subunit in an (~)a complex acted as an efficient acceptor for the energy absorbed by the trimer [191. In contrast, the complex ~ " :~,i~~AP~A~'..~ ~ Pa )'~.,OVV does not undergo subunit exchange. Of the energy absorbed by this complex, 85 % is emitted at 680 nm and the remainder originates from the allophycocyanin contained within this complex [21]. These results indicate t h a t the allophycocyanin B-homologous trimer (~Am~xP)a is an artifact of isolation. Structure o/the A N l 1 2 phgcobilisome. It was noted above t h a t two 18 S subassemblies containing allophycocyanin complexes 1 and 2 (fig. 8 D) were obtained per A N l l 2 phycobilisome. Two lines of evidence indicate t h a t each of the allophycocyanin complexes 3 and 4 is also present twice in the core: 1) a comparison of the compositions (and yields) of the complexes with t h a t of the intact A N l l 2 phycobilisome, and 2) the near-coincidence of the molar absorption spectrum of the phycobilisome with that generated by summing the spectra of the constituent complexes (fig. 8 D) taken in appropriate molar proportions [21]. A particularly fascinating feature of the structure of the phyeobilisome core is the finding that the allophvcocyanin monomer participates as a building block within four subcomplexes, each of different composition. Analysis of the 11 S fraction showed t h a t the (~D~.27,000 phycocyaniu complex was the major component along with small amounts of the (~A"$AI'h' 10,500 and ta ~a %A I ' B %AI)~PaA P ~)'~v,auu complexes. The structure of the A N l l 2 phycobilisome that emerges from these analyses is shown in figure 8 A. Two aspects of the proposed arrangement are arbitrary. The two core cylinders (fig. 8 B) are shown antiparallel to each other. A parallel arrangement has not been excluded. The order of the complexes within each core cylinder has not yet been fully established. The nearest-neighbor relationship of complexes 1 and 2 was established by their isolation within the 18 S particle. A larger particle containing complexes l, 2 and 3 (tig. 8 D) has been obtained from AN112 phycobilisomes by limited tryptic digestion and fractionation under appropriate conditions (D. d. Lundell, unpublished observations). However, the position of complex 4 (fig. 8 D) has been assigned arbitrarily on the basis of considerations of efficiency of energy transfer. STRUCTURE-FUNCTION

RELATIONSHIPS.

Spectroscopic properties o[ the rod subslruclures. As described above, the rod substructures are made up of phycocyanin-linker polvpeptide complexes, (~)~.27,000, (~)~'33,000 and (~)~-30,000, listed in order starting with the disc proximal to the core. The absorption and fluorescence emission spectrum of each of these complexes is different [22, 26]. The absorption spectra of the three complexes are shown in figure 9. The absorption spectrum of the (~)~.33,000 complex is red-shifted with respect to t h a t of (~)6"30,000, and t h a t of (~)6" 27,000 is, in turn, red-shifted relative to that of (~)~. 33,000. The influence of the various linker polypeptides on the spectrum of phycocyaniu can best be seen in the inset to figure 9, which shows difference spectra generated by subtraction of the spectrum of free phycocyanin from t h a t of each of the three complexes.

STRUCTURE OF A ~ SIMPLE ~) PHYCOBILISOME

175

The complexes show strongly enhanced and red-shifted absorbance above 600 nin relative to that of free phycocyanin, This is most pronounced for (a~),.27,000. The fluorescence quantum yield of the complexes is also much higher than that of the free protein [22]. As anticipated from the data in figure 9, the fluorescence emission spectra of the complexes follow the order (~)6"30,000, (~)6"33,000, (=~)6"27,000 going towards the red end of the spectrum (fig. 10). These spectra show that the organization of the rods leads to polar structures with respect to energy transfer. Calculations of the overlap integral J for the pairwise overlap of the emission spectrum of each donor with the absorption spectrum (a) of the proximal acceptor(s) show that the spectral shifts induced within the linker polypeptide complexes signilicantly favour the forward energy transfer rate along the rods towards the core and to the allophycocyanin in the core. A detailed quantitative analysis requires further spectroscopic studies. 400 t

I

F

I

I

I IlO

T

T

1

I

(PC,27K-PC~)

) zo

i

500

T

E

(_1

E

200

~ \ 0~,~ 'Pc30K-PC) '\ -I0 "-'-~~-[ ~, 580 600 620 640 660 680 !..~.. X(nm) -~

i O0

- P C. 2 7 K

pC,30K ~ kr~ P ~C . 3 3 K 600

620

640 660 Wovelength(nm)

680

700

Fu~. 9. - - Absorption spectra of phgcocganin-linker polgpeptide complexes. Absorption spectra of [(a~)8" 30,000]n, [(a~)6"33,000]n and (~)6"27,000 rod and disc phycocyanincontaining complexes were determined in 0.6 M N a K - p h o s p h a t e , p H 8.0. The inset shows difference spectra obtained by subtraction of the s p e c t r u m of free phycocyanin in the same solvent from t h a t of each of the three phycocyanin-linker polypeptide complexes. PC represents an (=~)~ h e x a m e r of phycocyanin; 30K, 33K, etc., represent polypeptides of Mr 30,000, 33,000, etc.

Spectroscopic properties o/the core domains. The absorption and fluorescence emission spectra of the core complexes are shown in figure 11 [20, 21]. The absorption spectrum of the arAP~AP ~3 c o r n -

176

A. N. GLAZER AND COLL. I

I

I

I

I

I

,ooI 80

\\

c 60 PC .30 K

o

PC.35K

k \\\ PC,27K

4o

20

I

600

I

l

620

I

I

I

I

640 660 Wavelength (nm)

I

680

I 700

FIG. 10. - - Fluorescence emission speclra of phgcocganin-linker polypeplide complexes. Spectra of the complexes (see fig. 9) were determined in 0.6 M N a K - p h o s p h a t e , p H 8.0. For abbreviations, see legend to figure 9.

plex 1 (fig. 8 D) is not included. Analysis of the spectrum of the 18 S particle and that of complex 2, ~2APnAP P2 " 1 8 , 3 0 0 9( 4 0 , 0 0 0 9 1 1 , 0 0 0 ) , indicates that the spectrum of complex 1 within the 18 S particle is the same as t h a t of complex 4, @p~2t,. 10,500 [20, 21]. The fluorescence emission spectrum of wild type [32, 33] and of A N l l 2 phycobilisomes [33] is the sum of two components centered at 680 and 660 nm, respectively, with the former being the dominant one. From these observations, and the spectroscopic data shown in figure 11, it is evident that the energy flow within the core follows the paths ~,~e

9

I 0 , 5 0 0 - ~ ,APB ~ AP~AI' p3 -10,500

t,

@v~;xp -+ @ v ~ P . 18,300.75,000

Overall aspeels of energy lransfer wilhin Syneehoeoccus 6801 pbycobilisomes. The phycobilisomc focuses excitation energy, on a small number of terminal acceptor chromophores. Analyses and electron microscopy show t h a t under normal culture conditions, the rods of Syneebocoecus 6301 phycobilisomes contain an average of 18 phycocyanin hexamers. Each phycocyanin monomer carries three phycoeyanobilins [14]. Hence, the rod elements contain a total of 324 bilins. Allophyco, cyanin [14] and allophycocyanin B [13] subunits, the M,. 18,300 polypeptide [20]-

STRUCTURE I

V

1200 I

O F A (( S I M P L E

I

l

]

I

1

I

I

I

(~AP/~AP) 2 .,s,3 4 O K % K !

A

1000

177

)) P H Y C O B I L I S O M E

100

(aAPf~AP)310.5K

l'/

I I I I I I I I

800 ~-

I

iI I

600

(a AP/~AP)310.5K /

E p

/

400 /

/

/

/ //

,\ \

\

\

\ \\'

((:1AP~AP)2.18.3.40K?I I K'~

~

7",,,

\ \,,, 0 -

560

600

640 X(nm)

680

610

650

650 670 X (nm)

690

FIG. 11. - - Absorption and fluorescence emission spectra of core subcomplexcs. Spectra of t h e c o m p l e x e s were d e t e r m i n e d in 50 m M N a K - p h o s p h a t e - - 10 % (v/v) glycerol, p H 7.0. A b b r e v i a t i o n s used are: 40 K* a n d 11 K* = t r y p t i c f r a g m e n t s of t h e Mr 75,000 polypeptide; all o t h e r a b b r e v i a t i o n s are defined in t h e legend to figure 8.

and the M, 75,000 polypeptide [23] each carry a single bilin. The energy from the rods is transferred to the 44 allophycocyanin and Mr 18,300 bilins within the core complexes (fig. 8) and from there to the four allophycocyanin B ~ subunit and M, 75,000 polypeptide bilins. This series of transfers may be represented schematically as follows: ROD SUBSTRUCTURE

Phycocyanin

CORE SUBSTRUCTURE

Allophgcocganin

Allophycocganin B

M~ 18,300 p o l y p e p t i d e

324 bilins

44 bilins

X..... 652 n m

F ~kmax 660 n m

F

-+

subunit 2 bilins M~ 75,000 polypeptide F

2 bilins

kma ~ 680 n m Ann. Microbiol. (Inst. Pasteur), 134 B, n ~ 1, 1983.

12

178

A. N, GLAZER AND COLL. Rt~SUMI~ CA

STRUCTURE

D'UN

PHYCOLIBISOME

(( S I M P L E

))

Cet article decrit les propridtSs des phycobilisomes d'une relative simplicite, ceux de Sgnechococcus6301 (Anacgstis nidulans). Les phycobilisomes du type sauvage ou de mutants vus (( de face )) au microscope 61ectronique prSsentent denx sous-structures de morphologies diffSrentes : d'une part, un centre, forms de deux objets contigus en forme de disques de 115 .~ de diametre; d'autre part, six batonnets formes chacun par l'empilement de plusieurs disques de 60 ~ d @aisseur et de 120 A de diametre, disposes en dventail autour du centre, le tout ayant une forme hdmidiscoidale. Chacun des composants du centre est forms de quatre disques de 30 s d'epaisseur. Chaque disque dans le M t o n n e t est un hexam~re de phycocyanine maintenu grace ~ l'interaction d'un polypeptide de liaison; sa composition est (~)~.X, off X reprSsente un polypeptide de liaison et ~f3 un monom~re de phycocyanine. Le disque le plus proche du centre est un complexe (~$)6"27 000. Une petite portion, M~ ~ 2 000, du polypeptide de Mr 27 000 est essentielle/~ la fixation de ce disque sur le centre. L'etude des phycobilisomes de cellules carencees en azote ou de mutants contenant une faible quantitd de phycocyanine par rapport ~ leur contenu en allophycocyanine a permis d'Stablir que le deuxieme disque du bfitonnet est un complexe (~)~-33 000. Des complexes (~)6"33 000 ou (af3)r 000 occupent, dans les b~tonnets, des positions distales par rapport ~ celle du disque (~)~. 33 000. Cette Stude ainsi que celle de la jonction centre-bfitonnet, a et6 facilit~e par l'isolement d'un mutant, A N l l 2 , qui poss~de des phycobilisomes dont les centres sont normaux mais dont les batonnets autour du centre ne comportent qu'un seul disque. La dissociation partielle de ces phycobilisomes incomplets, la sdparation et la caracterisation des sous-complexes resultants, a permis de dSterminer la composition de quatre complexes ~ trimeriques ), distincts, chacun 6tant prasent en deux exemplaires par phycobilisome. Cos complexes qui constituent le centre du phycobilisome ont la composition suivante : (~)AP.10 500 (Xma~ ~ 660 rim); ( k ~ , , ~ 6 6 0 n m ) , "(~h~P~xAPB~3AP) ' 1 0 5 0 0 (Xr'.... a 680 nm), off A v et ~tvB sont les sous-unitSs ~ respeetives d'allophycocyanine et d'allophycocyanine B eL ou est une sous-unit6 commune /~ ces deux biliprotieines; ( ~ ) ~ P - 1 8 3 0 0 , (40 000.11 000) (~m~ F ~ 680 nm) off les polypeptides 40 000 et 11 000 sont dSrivOs du polypeptide M~ 75 000 apr6s digestion tryptique. Les complexes (~)~tv et (~)~P.18 300.(40 000-11 000) oeeupent des positions adjacentes l'une de l'autre dans le centre. Les positions relatives des autres complexes restent h d6terminer. Les polypeptides de M~ 75 000 et 18 300 portent chaeun un chromophore de phyeoeyanobiline. L'interaetion de la phyeocyanine avec les polypeptides de liaison s'aceompagne d'un ddplaeement vers le rouge du spectre d'absorption de la biliprot~ine, d'nne augmentation de son absorbance aux longueurs d'onde dlevdes et du rendement quantique de fluorescence. Les maximums de fluorescence des complexes de phycoeyanine avee les polypeptides de M, 30 000, 33 000, et 27 000 sont situds respeetivement ~ 643, 648 et 652 nm. La migration de l'6nergie h l'int6rieur des batonnets est done prdf~rentiellement dirig~e vers le centre. La sequence du transfert d'~nergie h l'intdrieur des phycobilisomes de Synechococcus 6301 que l'on peut d~duire de ees 6tudes est la suivante : PC. 33 000 xAP > APB - ~ P C . 3 0 0 0 0 - + P C . 2 7 000: ? J/ PC .30 000 ~ 18 300 -+ 75 000 ~OTS~CLt~S : Phycobilisome, Ultrastructure, Assemblage, Migration de l'energie.

STRUCTURE OF A (( SIMPLE )) PHYCOBILISOME

179

ACKNOWLEDGMENTS This research was supported in part by grants PCM79-10996 (A. N. Glazer) and PCM80-10650 (R. C. Williams) from the National Science Foundation. D. J. Lundell was supported by postdoctoral fellowship GM07179-03 from the United States Public Health Service, and G. Yamanaka by a fellowship from the United States Public Health Service Training Grant T32 GM07232-04. A. N. Glazer thanks the John Simon Guggenheim Memorial Foundation for the award of a fellowship. REFERENCES [1] ALLEN, M. M. & SMITH, A. J., Nitrogen chlorosis in blue-green algae. Arch. Microbiol., 1969, 69, 114-120. [2] BRYANT, D. A., GUGLIELMI, G., TANDEAU DE MARSAC, N., CASTETS, A.-M. & COHEN-BAZIRE, G., The structure of cyanobacterial phycobilisomes: a model. Arch. Microbiol., 1979, 123, 113-127. [3] ELEY, J. H., Effect of carbon dioxide concentration on pigmentation in the blue-green alga Anacyslis nidulans. Plant Cell Physiol., 1971, 12, 311-316. [4] ENGELMANN,T. W., Farbe und assimilation. Bot. Ztg., 1883, 11, 1-13. [5] GANTT, E., Structure and function of phycobilisomes: light-harvesting pigment complexes in red and blue-green algae. Int. Reo. Cytol., 1980, 66, 45-80. [6] GANTT, E., Phycobilisomes. Ann. Rev. Plant Physiol., 1981, 32, 327-347. [7] GANTT,E. & LIPSCHULTZ,C. A., Phycobilisomes of Porphyridium cruentum. - I. Isolation. J. Cell Biol., 1972, 54, 313-324. [8] GANTT, E., LIPSCHULTZ,C. A. & ZILINSKAS, B., Phycobilisomes in relation to the thylakoid membranes. Brook. Syrup. Biol., 1976, 28, 347-357. [9] GANTT, E., LIPSCHULTZ,C. A., GRABOWSKI,J. & ZIMMERMAN,B. K., Phycobilisomes from blue-green and red algae. Isolation criteria and dissociation characteristics. Plant Physiol., 1979, 63, 615-620. [10] GLAZER, A. N., Photosynthetic accessory proteins with bilin prosthetic groups, in (( The biochemistry of plants ))(M. D. Hatch & N. K. Boardman) Vol. 8 (pp. 51-96). Academic Press, London, New York, 1981. [11] GLAZER,A. N., Phycobilisomes: structure and dynamics. Ann. Rev. Microbiol., 1982, 36, 171-196. [12] GLAZER, A. N., APELL, G. S., Hixsoi, C. S., BRYANT, O. A., RIMON, S. & BROWN, D. M., Biliproteins of cyanobacteria and rhodophyta: homologous family of photosynthetic accessory pigments. Proc. nat. Acad. Sci. (Wash.), 1976, 73, 428-431. [13] GLAZER, A. N. & BRYANT, D. A., Allophycocyanin B (~.... 671, 618 nm). A new cyanobacterial phycobiliprotein. Arch. Microbiol., 1975, 104, 15-22. [14] GLAZER,A. N. & FANG, S., Chromophore content of blue-green algal phycobiliproteins. J. biol. Chem., 1973, 248, 659-662. [15] GLAZER, A. N., WILLIAMS, R. C., YAMANAKA, G. & SCI-IACHMAN,H. K., Characterization of cyanobacterial phycobilisomes in zwitter!onic detergents. Proc. nat. Acad. Sci. (Wash.), 1979, 76, 6162-6166. [16] GOEDHEER,J. C., Spectral properties of the blue-green alga Anacystis nidulans grown under different environmental conditions. Photosynthetica, 1976, 10, 411-422. [17] GUGLIELMI,G., COHEN-BAzIRE, G. & BRYANT, D. A., The structure of Gloeobacter violaceus and its phycobilisomes. Arch. Microbiol., 1981, 129, 181-189. [18] LAU, 12. H., MACKENZIE,M. M. & DOOLITTLE,~'V. F., Phycocyanin synthesis and degradation in the blue-green bacterium Anacystis nidulans. J. Bacl., 1!)77, 132, 771-778.

A. N. GLAZER AND COLL.

180

[19] LUNDELL, D. J. & GLAZER, A. N., Allophycocyanin B. A common ~ subunit in Synechococcus allophycocyanin B (k.... 670 nm) and allophycocyanin (k~;~ 650 nm). J. biol. Chem., 1981, 256, 12600-12606. [20] LUNDELL,D. J. 85 GLAZER,A. i . , Molecular architecture of a light-harvesting antenna. Structure of the 18S core-rod subassembly of the Synechococcus 6301 phycobilisome. J. biol. Chem. 258, 894-901. [21] LUNDELL, O. J. 85 GLAZER,A. N., Molecular architecture of a light-harvesting antenna. Core substructure in Synechococcus 6301 phycobilisomes: Two new allophycocyanin and allophycocyanin B complexes. J. biol. Chem. 258, 902-908. [22] LUNDELL, D. J., WILLIAMS, R. C. 85 GLAZER, A. N., Molecular architecture of a tight-harvesting antenna. In vitro assembly of the rod substructures of Syneehococcus 6301 phycobilisomes. J. biol. Chem., 1981, 256, 35803592. [23] LUNDELL,D. J., YAMANAKA,G. 85 GLAZER,A. N., A terminal energy acceptor of the phycobilisome: The 75,000-dalton polypeptide of Synechococeus 6301 phycobilisomes - - a new biliprotein. J. Cell Biol., 1981, 91, 315-319. [24] MIMURO, M. 85 FUJITA, Y., Estimation of chlorophyll a distribution in the photosynthetic pigment systems I and II of the blue-green alga Anabaena variabilis. Biochim. biophys. Acla (Amst.), 1977, 459, 376-389. [25] MOBSCHEL, E., KOLLER, K.-P., WEHBMEYER, W. 85 SCHNEIDER, H., Biliprotein assembly in the disc-shaped phycobilisomes of Rhodella violacea. I. Electron microscopy of phycobilisomes in silu and analysis of their architecture after isolation and negative staining. Cytobiologie, 1977, 16, I18-129. [26] 0QUiST, G., Light-induced changes in pigment composition of photosynthetic lamellae and cell-free extracts obtained from the blue-green alga Anacyslis nidulans. Physiol. Plant., 1974, 30, 45-48. [27] REDLINGER, T. 85 GANTT, E., Phycobilisome structure of Porphyridium cruentum. Polypeptide composition. Plant Physiol., 1981, 68, 1375-1379. [28] TANDEAU DE MARSAC, i . 85 COHEN-BAZIRE, G., Molecular composition of cyanobacterial phycobilisomes. Proc. nat. Acad. Sci. (Wash.), 1977, 74, 1635-1639. [29] WANG, R. T., STEVENS, C. L. R. 85 MYERS, J., Action spectra for photoreactions I and II of photosynthesis in the blue-green alga Anacystis nidulans. Photochem. Pholobiol., 1977, 25, 103-108. [30] YAMANAKA,G. 85 GLAZER,A. N., Dynamic aspects of phycobilisome structure. Phycobilisome turnover during nitrogen starvation in Synechococcus sp. Arch. Microbiol., 1980, 124, 39-47. [31] YAMANAKA,G. 85 GLAZER,A. N., Dynamic aspects of phycobilisome structure: modulation of phycocyanin content of Synechococcus phycobilisomes. Arch. Microbiol., 1981, 130, 23-30. [32] YAMANAKA, G., GLAZER, A. N. 85 WILLIAMS, R. C., Cyanobacterial phycobilisomes. Characterization of the phycobilisomes of Synechococcus sp. 6301. J. biol. Chem., 1978, 253, 8303-8310. [33] YAMANAKA, G., GLAZER, A. N. 85 WILLIAMS, ]2~. C., Molecular architecture of a light-harvesting antenna. Comparison of wild-type and mutant Synechococcus 6301 phycobilisomes. J. biol. Chem., 1980, 255, 11004-11010. [34] YAMANAKA,G., LUNDELL,D. J. 85 GLAZER, A. N., Molecular architecture of a light-harvesting antenna. Isolation and characterization of phycobilisome subassembly particles. J. biol. Chem., 1982, 257, 4077-4086. [35] Yu, M.-H. 85 GLAZER, A. N., Cyanohacterial phycobilisomes. Role of the linker polypeptides in the assembly of phycocyanin. J. biol. Chem., 1982, 257, 3429-3433. [36] Yu, M.-H., GLAZER,A. N. 85WILLIAMS,R. C., Cyanobacterial phycobilisomes. Phycocyanin assembly in the rod substructures of Anabaena variabilis phycobilisomes. J. biol. Chem., 1981, 256, 13130-13136. -

-