Immunogold localization of photosystem I and photosystem II light-harvesting complexes in cryptomonad thylakoids

Biol Cell (1992) 74, 187-194 © Elsevier, Paris

187

Original article

Immunogoid localization of photosystem I and photosystem II light-harvesting complexes in cryptomonad thylakoids Christiane Lichtl6 ~, R Michael L McKay 2, Sarah P Gibbs 2 *Laboratoire des Biomembranes et Surfaces cellulaires v~gdtales (UA CNRS D 0311), Ecole Normale Supdrieure, 46, rue d'Ulm, 75230 Paris Cedex 05, France; 2Department o f Biology, McGill University, Montrdal, Quebec H3A 1B1, Canada (Received 22 April 1991 ; accepted 17 December 1991) Summary - The molecular organization of the thylakoids of Cryptomonas rufescens was studied by immunoelectron microscopy

employing antibodies against photosystem (PS)-I and two PS-II antenna proteins. The PS-I complex and the 19-kDa chlorophyll a/c light-harvesting (LH) protein are both localized along the length of the thylakoid membranes. The external membranes of the paired thylakoids are enriched in PS-I whereas the chlorophyll a/c LH protein is more concentrated in the internal or appressed membranes. However, unlike the situation in higher plants and Chlamydomonas, there is not a marked asymmetry in the concentration of PS-I and chorophyll a/c LH protein in the two types of membranes. Double labelling studies of sections and isolated PE-PS-II particles with anti-phycoerythrin and anti-LH confirmed that phycoerythrin is localized in the thylakoid lumen and that this pigment exists in two forms, a fraction closely associated with the thylakoid membranes and another fraction free in the lumen. These results confirm the uniqueness of cryptomonad thylakoids.

Cryptomonas rufescens I light-harvesting protein / photosystems / phyeoerythrin / thylakoids

Introduction

Materials and m e t h o d s

The organization of the photosynthetic apparatus of cryptomonads is unique among the chromophyte algae [36, 37]. In addition to possessing chlorophyll (chl) c, cryptophyte chloroplasts have phycobiliprotein localized in the thylakoid lumen [6, 8, 15, 35]. Both pigments, chl c and phycobiliprotein (phycoerythrin (PE) or phycocyanin depending on the algal species) form antenna pigment-protein complexes which independently transfer light energy to photosystem (PS)-II [4, 17]. Treatment of isolated thylakoids with detergent and separation on sucrose gradients has allowed the isolation of these two antenna complexes, the chl a/c antenna [13, 19, 24] and the PE-PS-II antenna [19]. Immunocytochemistry has confirmed the localization of PE inside the thylakoid lumen [20, 22, 28, 29] and the association of the chl a/c antenna with the thylakoid membranes [24]. However, it is not known whether there is a differential distribution of PS-I and PSII on appressed and non-appressed thylakoid membranes of cryptomonads as there is in green algae and higher plants. In this study we have employed immunoelectron microscopy to determine the distribution of PS-I and of a PS-II-associated chl a/c antenna protein on the internal (appressed) and external (non-appressed) membranes of the paired thylakoids. We also confirm that the discs attached to isolated inside-out thylakoid vesicles of the PEPS-II antenna fraction [19] are indeed phycoerythrin.

Culture conditions

Abbreviations: chl, chlorophyll; CP, chlorophyll-proteincomplex; GAM, goat anti-mouseimmunoglobinG; GAR, goat anti-rabbit immunoglobin G; LH, light-harvestingprotein; PE, phycoerythrin ; PS, photosystem.

Cryptomonas rufescens Skuja cells were grown in S2T2 medium at 17°C as previously described [16]. Cells were harvested late in the logarithmic or early in the stationary phase of growth. Chloroplast fractions and phycoerythrin extraction The isolation of the PS-II antennae (fractions 2 and 3) and of PS-I (fraction 1) on a sucrose step gradient was performed as described by Lichtl~ et al [19]. The polypeptides of whole thylakoids and of the different fractions were separated on polyacrylamide slab gels [14] as described previously [19]. The phycoerythrin was partially extracted from thylakoids of algae placed for 30 min at 4°C before centrifugation (1500 g) [15].

A ntisera preparation Phycoerythrin antiserum raised in rabbit against PE 566 from Cryptomonas ovata [10] was the gift of R MacColl (New York State Department of Health, Albany, NY). Antiserum to PS-I raised against chlorophyll-protein (CP) complex I of spinach was the gift of B Lagoutte (CEA, Service de Biophysique, Saclay, France). Spinach CPI was isolated according to Setif et al [26] and 50 tsg was mixed with Freund's adjuvant and injected into a mouse. A second injection was made three weeks later. Antiserum to the 19-kDa protein of the chl a/c fight-harvesting complex (LH) of C rufescens, designated LH-antiserum, was prepared as follows. The polypeptides of fraction 2 were separated by gel electrophoresis and then transferred to a nitrocellulose sheet [32] and stained in Ponceau red to determine the exact position of the polypeptides. The 19-kDa band was cut out, ground in liquid nitrogen, mixed with Freund's complete adjuvant, and injected into a rabbit. Four injections were given at weekly intervals and the serum tested from the third week on. Preimmune serum was taken before the first immunization.

c Lichtl~ et al

188

cles, or goat anti-mouse (GAM), l0 nm gold particles (Janssen Pharmaceutica, Beerse, Belgium). For the double labelling experiments, the dilutions were the same as for single antiserum. The first labelling was made with PE-antiserum and GAR 5 nm, and the second labelling with LH-antiserum and GAR l0 nm. Improved contrast of the membranes was achieved by overnight exposure to OsO~ vapors followed by 2°70 uranyl acetate staining. Sections were viewed in either a Philips EM 300 or a Philips EM 410 electron microscope operated at 80 kV. The following control experiments were performed: incubation in immunoglobulin G alone omitting the antibody step ; incubation with non-immune serum in place of the antiserum ; and incubation with PBS plus 207o BSA in place of the antiserum.

Immunoblotting The separated polypeptides of whole thylakoids, fractions 1 and 2 were transferred electrophoretically to nitrocellulose sheets [32]. After blocking with 307o bovine serum albumin (BSA) in TBST (50 mM Tris, pH 7.4, 150 mM NaCl, 0.1070 Tween-20) for 1 h, blots were incubated 2 h at room temperature in the anti-LH serum or in the anti-PS-I serum diluted with TBST plus 0.3070 BSA at different dilutions from l:10 ~ to l:10~. The blots were washed several times with TBST and then incubated for 45 min in a 1:5 x l0 ~ dilution of horseradish peroxidase-conjugated goat anti-rabbit for anti-LH or goat anti-mouse immunoglobulin G for anti-PS-I (Bio-Rad, Paris, France) in TBS. After several washes in TBST, immunoreactive bands were visualized by incubating blots in the substrate paraphenylene diamine pyrocatechol in 50 mM Tris, pH 7.4, plus 0.02070 H20 2.

lmmunocytochemistry on negatively stained preparations Fraction 3 was removed from the sucrose density gradient and placed on formvar-coated nickel grids as described by Lichtl~ et al [19]. The labelling with antiserum was performed according to the method of Vallon et al [34]. Grids were floated on drops of anti-LH 0:250 dilution in PBS plus 2070 BSA) for 30 min or anti-PE 0:500 dilution in PBS plus 2070BSA) for 30 min, followed by GAR 10 nm for 20 min. For the double labelling, the antisera dilutions were the same and the grids were exposed successively to anti-PE and GAR 5 nm and to anti-LH and GAR 10 nm. The grids were post-stained 5 min with 2°7ouranyl acetate.

Postembedding immunocytochemistry Cultures of C rufescens were grown to a density of 7.0 x 10s cells/ml and harvested by gentle centrifugation. Cells were fixed at 4°C for 90 min in 1.0070glutaraldehyde in 0. l M sodium phosphate buffer, pH 7.4. Following three 10-min rinses in buffer alone, the cells were dehydrated in a graded ethanol series at 4°C and embedded in LR White medium grade resin (JB EM Services, Montr6al, Qu6bec) by the following protocol: 100O7o ethanol/resin, l : l , two changes at 4°C, 30 min and 1 h; pure resin, three changes at room temperature, 45 min, overnight and 2 h ; pure resin at 58°C for 20 h. Pale gold-coloured sections cut with a diamond knife were collected on formvar-coated nickel grids. Grids were floated successively on drops of the following solutions: phosphate-buffered saline (PBS), 10 min; PBS plus 207o BSA, 15 min; antisera diluted in PBS plus 207o BSA (LHantiserum, 1:250 dilution, overnigth ; PE-antiserum, l : l0 ~ dilution, for l h; PS-I-antiserum, 1:250 dilution overnight). After washing with PBS, the sections were placed for 1 h on drops of gold-labelled secondary antibody diluted i:30 in PBS plus 2°7o BSA either goat anti-rabbit (GAR), 5 nm or 10 nm gold parti-

L "

Quantification of labelling The thylakoids of cryptomonad chloroplasts are loosely associated in pairs, each pair thus has two external and two internal membranes. For the two antisera raised against membrane proteins, anti-PS-1 and anti-LH, gold particles located partially or entirely over the external membrane were counted as labelling the external membrane (category 1, see fig 4). Gold particles lying between the two dense lumens of a thylakoid pair were counted as internal membrane labelling (category 2, fig 4). Particles lying over the dense thylakoid lumen (category 3, fig 4) were count-

,

: ;

,

,

/

i ii

,

i

94 67 43

k..~J ~.,- ",,,~

30

•

-.:

.,

..

20.1 A

14.4

.J: .... "

....

Fig 1. Li-PAGE profiles and immunoblots of thylakoid fractions from Cryptomonas rufescens. Lane A : Li-PAGE profile of polypeptide components of PS-I (fraction 1, see Materials and methods) ; lane B : immunoblot of A probed with PS-I-antiserum from spinach ; lane C : Li-PAGE profile of polypeptides associated with PS-II (fraction 2) ; lane D : immunoblot of C probed with LHantiserum; lane E: Li-PAGE profile of polypeptide components of whole thylakoids; lane F: immunoblot of E probed with PS-Iantiserum from spinach ; lane G : immunoblot of E probed with LH-antiserum. Protein markers from Pharmacia kit : ph0sphorylase, 94 kDa; bovine serum albumin, 67 kDa; ovalbumin, 43 kDa; carbonic anhydrase, 30 kDa; soybean trypsin inhibitor, 20.1 kDa; ~-lactalbumin, 14.4 kDa.

000Z01 × '. tort ~'0 = Jeff "(nl) u~tun I p!o~lel,~qi ~suzp ~ql J~AO p~le301 s~13!lJed ' [ '. J!ed p!o)lel~ql e JO S~tll3Jqttl~tU ll~tla~ltl! ~ql J~AO 9~1e301 s~p!laed 'Z '. a!ed p!o~lel,~qi e JO (ztu) ~ueJqua~tu leUJZlx~ ~ql J~^O p~le3o I szl3!lJed ' I : s~!JO~le~ ~zaql o1 p~u~!sse z J ~ a s ~ p •!lJed plOD "P~llZqelun s! (s) etuoals ~ql se~J~q~ P~lI~qel ~l!^ezq ~Je sp!o)lel~ql ~ q l "suassafnJ 0 j o I I - S d JO u!zload H q eO>l-61 ~ql isu!e~e p~s!eJ tuna~s!lue ql!m P~ll~qeI tlO!lD~s lSeldOJOlqS) "I~ "000E01 x '. m r / i Y 0 = Je~l 'a!ed J ~ q l o u e j o (ztu) s~ueaqtu~tu ieua~lx~ 9~1 ~q] ~ae se pzle3!pu! ~Je aued p!o)lel~q:l e j o (nl) su~tun I oa~l ~qj. 'p~^J~sqo ~Je s~13!laed plo~ oN '(IoJluo3) plo~ tuu 01-'ttVD ,~q 9 ~ O l i O ] t u n a , s ~untuuat~ad q l ! ~ P~llZqel lseldOJolqs) "£ '000Z01 × '. t u r / Z ' 0 = ae~l 'J!ed p!o)lei£ql e ] o su~tun I ' n I '. a]ed p!o)lel£ql e IO s~ueaqtu~ua leUJZlx~ '~uJ "P~ii~qelun ~ae (I) s~Inqol ~t P!d!l p u e (s) etuoJls lseldoJolq~ ~ q / "sp!o>leI~ql ~ql J~AO pZZ!Ie301 zae S~13 •!laed plo~ ~ q I "plo~ tuu 01-1AIVD ql!,~ p~)lJetu p u e q3eu!ds tuoaJ tuna~s!lue-l-Sd ql!m P~ll~qel suassafnJ 0 j o lseldOJolqs) "1~-Z s~!:I

....

" '

"

.

"

.-.-.

'-

-

~ . . . =

~

. " ' ~ ' ~ . , ~ , , ~.'k"~';'~i',-"-.:.,,,~..'r.~_~,:~.~" -~

-.~-;,.~.~

--~. " - - . . -

....... ,,i" '..

.

.

,~--.:; .;

~,4_~.

~'..'-"

"

• "

-

~ ' ~ u ~ . - , ~

..":,~-..-"'.'r'.t,'~

""-~ ~..~-..--

.

'

-.

.,

.

. .. " : ~

&..-

. .~.~ ""~;.~

"

~

.~ll~.. ~

~-~'-

.... ~ ~ , ~ , . ~ , . ~ , ~ ' - =

.... , "

~ "

~

.....

~

~--"

:

".." ~

'

":," . ' ~:"

~ .~

. " .

'

~ .-

",c Z .

.

~ .

.,"~""~',, .

~ .-

.~, ,~..

.

.

,,

-

,

-

......

I~l].

"

,

~

~

• :.:;.~ ~.~

.

'

" •

.~:...,.f~

.

.~---~.-'~ . - ,- - . . ~ ' - ~ , ~ . d . •~ . '- ~ k ~ - ' ~ ".: ':; '~- ~-.~7 .- "~" % . ~~ .- - , ~ , ~ . - ~ ,-" ~, • - ~

-

.~ ~ ' ~ . . o . , .

,..

-7,

. . . . . . . . . . . . . . . . . .

~

-- .-, : = ..

,

.

~

%

" . ~ ~ : - , ~~

..

~

"

.: ~.

'~ ~ ~

_,<-'h.~ ~ -

~

~

"" ~ .~"~_-~., ,

~d

...

.

.k

"~

"

+-

'~ ? t !i

190

c Lichtl~ et al

ed separately and then half were arbitrarily assigned to the external membrane and half to the internal membrane. For antiLH, 3102 gold particles were counted; for anti-PS-I, 1877 particles were counted.

PSi

LH -o.o.eI !

-S 5 0 -

OOll OOOl OOOl OOOl OOOl OOOl OOOl OOOl OOOl ooei ooei oool ooo| ooe| oooi ooen OOOl oool ooei ooe i

Results l

Immunoblotting The proteins associated with the PS-I of C rufescens (fraction 1 on a sucrose gradient ; fig 1, lane A), those of the chl a/c antenna (fraction 2 on a sucrose gradient; fig 1, lane C) and those of the whole thylakoids (fig 1, lane E) were separated on polyacrylamide gels and stained with Coomassie blue [19]. After immunoblotting, anti-LH recognized exclusively the 19-kDa protein of the chl a/c antenna (fig 1, lanes D and G), whereas anti-PS-I of spinach reacted with a 67-kDa protein corresponding to the PS-I centers of fraction 1 (fig 1, lane B) and whole thylakoids (fig 1, lane F). No reaction was observed with preimmune sera (not shown). Ludwig and Gibbs [20] have shown by immunoblotting that anti-PE 566 of C ovata cross-reacts with the/3 subunit of PE 545 of the cryptophyte alga Rhodomonas lens.

~

_

-

000 000 000 000 000 000 000 000 000 000 000 000 000 000 000 000 000 000

30-

o

c-

010'L

13..

ext

int

• o, i

ext

int

Fig 5. Distribution of gold particles over the external (ext) and internal (int) membranes of the paired thylakoids of Cryptomonas rufescens. Sections were labelled either with antiserum to PS-I of spinach or with antiserum to the 19-kDa chl a/c LH protein associated with PS-II in C rufescens.

Immunolabelling of sections The thylakoids of Cryptomonas rufescens are associated in pairs and in cells grown in low light, the pairs are frequently piled up to form large stacks which fill most of the chloroplast (figs 2 - 4 ) . After fixation in glutaraldehyde and embedding in LR White and in the absence of OsO 4, the PE-containing thylakoid lumens appear electron dense, whereas the thylakoid membranes appear electron translucent (figs 2 - 4). Even within the large stacks, it is easy to identify the thylakoid pairs, for a space of approximately 4 - 7 nm separates the two dense lumens of a pair, whereas a larger space ( 1 0 - 1 5 nm) separates the dense lumens of adjacent pairs. In these preparations, the two internal membranes of a thylakoid pair appear as a single electron translucent area, whereas a thin dense line can be seen separating the two electron translucent external membranes of adjacent thylakoid pairs. In a section of a chloroplast labelled with antibody to PS-I ofcspinach, gold particles are distributed along the length of the thylakoids (fig 2). Very few are observed over the chloroplast stroma or over the pyrenoid (not shown). Control sections labelled with preimmune serum or by incubation in PBS alone followed by gold-labelled secondary antibody showed no labelling (fig 3). To quantitate the distribution of PS-I labelling between the external and internal membranes of the paired thylakoids, regions where paired thylakoids had been cut perpendicularly were marked at each end with vertical lines and all gold particles between the lines counted and categorized. Thus the length of external membrane analyzed exactly equaled the length of internal membrane analyzed. Of the 1877 particles counted, 42.6°70 were localized over the external membranes, 29.6°70 over the internal membranes, and 27.7°70 were localized over the lumens. Since PS-I is a membrane localized, the lumenal counts were arbitrarily assigned half to each membrane. Figure 5 shows that anti-PS-I labelling was slightly higher (56.5°70) over the external membranes than over the internal membranes (43.5070). In a chloroplast section labelled with antiserum prepared against the 19-kDa chl a/c antenna protein of PS-II of C

rufescens, the chloroplast thylakoids are highly labelled whereas the chloroplast stroma is unlabelled (fig 4). Particle counts showed that 19.5070 of the anti-LH labelling was over the external membranes and 32.2°70 was over the internal membranes. Taking into account the particles localized over the lumen (48.2070), the L H protein is more concentrated on the internal (54.2°70) than on the external (45.8°70) membranes of the thylakoid pairs. In a chloroplast which has been double labelled with anti-PE marked with 5 nm gold followed by anti-LH marked with 10 nm gold, the thylakoids are heavily labelled with each antibody (fig 6). A quantitative analysis of anti-PE labelling (3004 particles counted) showed that 13.7°70 of the particles were located over the external membrane not touching the dense lumen, 21.7°70 were over the external membrane touching the lumen, 34.9°70 were over the lumen, 22.9°70 were over the internal membrane touching the lumen and 6.9°70 were over the internal membranes not touching either lumen. Ludwig and Gibbs [20] have demonstrated in another cryptomonad species, Rhodomonas lens, that all PE is inside the thylakoid lumen and that even those gold particles located over the membrane mark lumenal PE. They concluded that much of the lumenal PE is membrane bound. Our data demonstrate that much of the PE is also membrane associated in C rufescens (65070 of the labelling) and furthermore that PE is associated with both the internal and the external membranes of the thylakoid pairs. In a cell of C rufescens from which PE has been partially extracted [15], PE has been preferentially extracted from the center of the thylakold lumen and regularly spaced rod-shaped structures are clearly seen attached to the inner (star) or outer (arrows) membranes of the thylakoid pairs (fig 7). Immunolabelling of &olated PE-PS-H particles Lichtl6 et al [19] isolated and characterized a photosynthetically active PE-containing subchloroplast fraction

Organization of cryptomonad thylakoids

191

Figs 6, 7. Double labelling of a chloroplast section with anti-LH marked with 10 nm gold and anti-PE marked with 5 nm gold. The small gold particles marking PE are observed both over the dense thylakoid lumen (lu) and the thylakoid membranes (me). The latter labelling is presumed to arise from membrane-associated lumenal PE. Bar = 0.2 gm; x 102000.7. Chloroplast of a cell of Crufescens from which PE has been preferentially extracted. Periodic rod-shaped structures are present in the lumen of the paired thylakoids. The rods may be attached to either the internal (star) or outer membrane (arrows) of the paired thylakoids, me, external membranes of a thylakoid pair; mi, internal membranes of a pair. Bar = 0.2/~m; x 150000.

(fraction 3) from C rufescens which is highly enriched in PS-II reaction centers and relatively deficient in LH chl molecules. In the electron microscope, these purified PEPS-II particles consisted of small vesicles 25 - 40 nm in diameter to which were attached small discs 1 0 - 12 nm in diameter and 6 nm thick. At places rods of 3 - 4 stacked discs were observed attached to the vesicles. Lichtl6 et al [19] interpreted the vesicles to be inside-out fragments of thylakoids with attached discs of phycoerythrin. To prove this, we have isolated these PE-PS-11 particles (fraction 3) and labelled them with anti-LH and anti-PE or double labelled them with both antisera prior to negative staining. When fraction 3 was labelled by antibody against the 19-kDa LH protein of the thylakoid membrane followed by GAR-10 nm gold, the gold particles lie over the membrane vesicles (fig 8a). When fraction 3 was labelled with anti-PE marked with 5 nm gold, the labelling is associated with the small 10-nm discs which are often arranged in rows (fig 8b). No labelling was observed when the antisera were omitted (fig 8c). When fraction 3 was double labelled by anti-LH marked with l0 nm gold and anti-PE marked with 5 nm gold, the large gold particles mark the membranes of the vesicles and the small gold particles the associated discs (figs 8 d - f ) . We conclude that the vesicles are indeed inside-out fragments of thylakoid mem-

branes with the 19-kDa chl a/c antenna protein located in the membrane and PE localized in the attached 10-nm discs.

Discussion In higher plants, PS-I is localized almost entirely on the membranes of the stroma thylakoids and on the limiting membranes of the grana stacks, whereas PS-II is localized largely on the appressed membranes of the grana stacks [1, 2, 5, 30, 33, 34]. A similar distribution of PS-I and PS-II components has been found in the green alga Chlamydomonas reinhardtii [33,34]. However, almost nothing is known about the distribution of PS-I and PSII in the chl c-containing chromophyte algae. In Chlamydomonas and in various other green algae, granalike configurations of the thylakoid membranes are observed [9], but in the chromophyte algae, the thylakoids are organized into extended lamellae frequently traversing the length of the chloroplast. Each lamella consists of three appressed thylakoids, except in the cryptomonads where the lamellae consist of paired thylakoids. Freezeetch studies of brown algae [3], dinoflagellates [31] and cryptomonads [7, 18, 23, 27] showed that the thylakoids

e

.: ....

•

"

' ",

"~ ~

., I'?.: .~ ~,

..,: -,; : ~,'..k ~.~-..

.,~,:, ~ :,"

":" #

:.'~ "4.-.~. ; ' r ~ , " -

:" -.v~"~" :.,

"

"

-

"

. ,~";.

~"-"

~~'-". ," ~ ~

"~

, .

v

~L~,:.~;,.. ~

'

~

'.~ r :

..", "

,,~ ~,.~,,,~ : . ,

~

.~

"

, ' ~ 3 ~ E ~ * _ "

"

,

, ".. ~

:'~.:~

=-.

,

•

----.

.~ ~q:-~l:-

": . - ' . ~ ' , . ; . . Z - ~ , _ ~ . ' :

-

:~';~'.~, ,-~.~._t'- ""

" "~

"'.-"- •

~"

....

. ~.:I *%

~X',~,.~-~":~

• . ":; ~ " P ,

,

~.:.,~..

r,. . . . .

.

-.h.-?'~.-~.~<~ ...-:..~.;._~ ~ , - ~ ~ ~ :

"i:;:~"

-

~.

. .....,..

,.,

., ,..:,

:

.,-.

.-~,

.~:

~ :~.~ .

~ - i,I," ,j;..:~k~,,~.-:,-~.,h

" :V

~ ' ~ " ~

_~'.

~..•.

.

~'.~

. ~.~..~

~'" "~

~

;-..-

"-

'

-.'

-~--:'~'~,'~,,:~

,¢'~-y~.~,- " - ' ,

:.~ ,"

•

.. - .

-" ~ ' - c ' ~ ' ~

"~,~"

,"

~. . , "

;

"...".

•

;~"

: -.•'.

..,

.

~-

.

•

:~,.

.¢~.

~ ......

_ _'~,~' .,

~ ,-'~..

~'-.~.-.~

.~

,

.~

,*,.,

";

•

.,':

.,.

-....,'.'::.~, ~ ",',T~ ~;,

,~

";".

.

:. ; ~ ~ , a . , , ~ . ~

~;~.,!._

.';~ ':" ,.~,. ~

..

• .~,,~,'.~.'::~.;..'-::

',

"

"".:"

i

.

" '~ ., " ..

!.-.~.:,.

. ~r. -.:

,..-,

" .~,

.,~

" ; , I ~ ' - ~

-~

:

-

:, ,.

. , . .

,~-i

~.¢.Z.'--~,L:," . . . . - ,. •

~,'7~;~?r"

,,. ,

"~

..

.,~ . ; ~

.-

_

" "

..-

;.,

, :.."• Z•" T,

.,

. .. '

~:.

-~.

'.." 'I" V,:," ." "

- •.',~.

.

"-,..-

-.

.'.~- ,~" .~ ~--~.'~ " Z ' : : ' ~

' ~"~I~,,,

o,

" '- "

~. ,

.~,"

•;.

.

"

, ". •

~ _

~,','.~.'_~-'..~.."L~.~_~ "

~".~&.~-..:b "' i!'..~':.:,"- ." "

. .

..~.

"

i " . "' : ' ~ ' , ~ - ~ . : "

..

.

."

"-..'L%..,: ~-,"-'-7..~ • k;::.~.

'. '~ " ~ ~ : : ", ~'-. ~::.~-': -. ~\z'm.~--.'" ~ : ~ - , - ' ~ ' ~ : ~--,, .: ,'~'~.~:-~. ,-.,, ~.~:~ .:.. <

~~

~ .

~.~:_.~.v~,~.~.~.,..

.:

"

,.

_~..~ ..- ( ,, .-(-~

-.

' .'r ,,

•

- - •,. ~.~:.~.: - ~~5,~ , ,--.-~.-.~ ~.~,.~ .

"';.r

, ,

: -"~i ~ -" ", ," . ";2~':4: .~ ~ ~,..~:. . ~ "~'"..-F~. -,,., .%,.~ .

&.'.: :. -,

.

/ - :.

~ - ~

,

~-

d l.;"-- ~.

.

..

"'

",

/.'.,..~•~.~o ,. ~;'~." .~

~.

"-

,,.~.

" C.-"~-'.,'," ~"i .I~:

•

-..

.: ':~"'-,~.r

- : ~,~.": ",.%'#11 •

~..,

~-

".~,-....~~.-,,:-..~.~' ,, '

~'.~'~'~"

).:

~,.

,~,'""~" ,

,

.-~,k~E,.~",~

.'. .~'I~-'~'~.~.~,~ • '- - ~

,~,~ ,~,~.~ ~,p---, ,~;~.~i~',~-~.~,~--. .. -..-

; ,

• ", ,,.~,.

. •

, .,~. ~ r . ~ . , , ' . . ,

,~"~

.--

.,,':,'

...,,~, ,•

.

~,

•

.~

" ..

.

.

,

, .

....

. . . . . . .

~ " ~

,

+ - :"

......

-.

,~,i~,.~.

~,

,

•

~:

-,,-"?'~IL~,':..•.

...~'~I~., ,

.

,y

~~.~.-,~.~ ~ : ~ . , ; . ~ , .

.....: ~.,.. ~--~. . . .

. .,.,:,.,.,..~:~,..

. ~,. ~ ,~

'

" •

.

.

.

" ; '" ~'

'

~" •

., " " .

~ .... " .... " ~ '

"."

"...... .,..

....

...

•

,,

:':.•

.

.!.,b~.. J ) L' "o~-

"

'

'e"!'.'.l_'./-~z~,

....

.r ~-, '.",~,~mm~,.,

~'.,~;.-~i~ '.~

.,-..~,-..

Fig 8. Negatively-stained isolated PE-PS-II particles (fraction 3) from C r u f e s c e n s , The particles consist o f small vesicles (v) to which small 10 nm discs (pe) are attached• a. Particles labelled with a n t i - L H a n d m a r k e d with G A R - 1 0 nm gold• The gold particles (long arrow) are located over the small thylakoid vesicles• b. Particles labelled with a n t i - P E a n d m a r k e d with G A R - 5 nm gold• The small discs attached to the vesicles are labelled (short arrow)• e. Control• Both antisera were omitted• d - f . Particles double-labelled with anti-LH (GAR-10 n m gold) and a n t i - P E (GAR-5 nm gold)• The large gold particles are localized over the thylakoid vesicles (long arrows) and the small gold particles (short arrows) mark the small discs a t t a c h e d to the vesicles• Bar = 0 . 2 / ~ m ; x 120000.

Organization of cryptomonad thylakoids of these chromophyte algae possess EF fracture faces resembling the EF fracture faces of the appressed and nonappressed thylakoid membranes of higher plants. This suggests that PS-II particles might be preferentially located on the appressed membranes of the thylakoid lamellae, but this has not been directly demonstrated. In this study we show by immunoelectron microscopy that PS-I is slightly more concentrated on the external membranes of the thylakoid pairs, whereas the 19-kDa chl a / c L H protein associated with PS-II is more concentrated on the loosely appressed internal membranes of the pair. First, however, the limitations of the immunolabelling technique must be discussed. In particular, we must ask if the resolution of the technique is sufficient to distinguish labelling on the external membranes from that on internal membranes. In our fixed tissues, the thylakoids are only 20 nm wide. An antibody molecule is approximately 8 nm long [25] whereas the gold particles are 10 nm in diameter. The maximum distance the center of a gold particle could be from the labelled antigen is 8 nm plus 8 nm (the secondary antibody) plus 5 nm (ie, 21 rim). Thus a gold particle localized over on the external membrane could sometimes have originated from an antigen in an internal membrane and vice versa, but because of the paired nature of the thylakoids, these errors should cancel each other out. A bigger problem is the large number of gold particles located over the lumen (28°7o in the case of PS-I labelling and 48070 in the case of anti-LH labelling). We arbitrarily assigned 50% of these labels to the internal membrane and 50070 to the external membrane. This procedure would make the labelling of the two types of membranes more equal than it actually is. If we simply discard the labels not definitely located on one membrane or another, we find that with anti-LH, 62.3% of the gold particles are over the internal membranes and with antiPS-I, 59.0070 of the labelling is on the external membrane. Thus it appears that there is a genuine difference between the labelling observed with anti-PS-I and with antiLH. However, compared with the situation in higher plants and green algae, it is clear that both PS-I and the 19-kDa chl a / c L H protein are found on both internal and external membranes and are distributed along the entire length o f the thylakoids. It is not clear how this relates to the freeze-etch studies, although Staehelin [30] showed that in cryptomonads, areas of concentrated EF particles may occur in the same membrane as areas of sparsely distributed EF particles. If these EF particles are PS-II reaction centers with associated chl a / c L H protein, then our data indicate that such patches of lateral heterogeneity would be present in both external and internal membranes. In higher plants, the chl a / b L H protein is believed to play a role in membrane stacking [2, 12, 21]. SpearBernstein and Miller [29] have recently proposed a model for crypt9monad thylakoids in which the chl a / c LH protein is located only in appressed internal membranes. Our data contradict this model for although anti-LH labelling is slightly more concentrated on internal membranes, it is also present on the external membranes. Also cryptomonad thylakoids are not tightly appressed as they are in higher plants, but have a 2 - 4 nm space between them, so true stacking may not exist. Our results with anti-PE labelling confirm the results of previous workers [20, 22, 27, 28] showing that PE is localized in the lumen and that a part of the PE is preferentially associated with the thylakoid membrane. In addition, we demonstrate for the first time that PE is associated with the internal and external thylakoid membrane. Since

193

PE transfers light excitation energy to PS-II [4, 17], this observation, like our anti-LH labelling results, indicates that PS-II is located on both membranes. We also show that when free PE is extracted from the cells, one sees small rods attached to the internal or to the external membrane. By immunolabelling negatively-stained preparations of a fraction containing the PE antenna attached to PS-II, we have confirmed the earlier suggestion of Lichtl~ et al [19] that the small 10-nm subunits associated to the membrane vesicles are discs of PE attached to inside-out thylakoid vesicles. These results support the observations of Hiller and Martin [1 l] that PE of cryptomonads has multiple forms, part of the pigment having a light-harvesting role whereas the other part associated with the membrane would transfer collected light energy to the PS-II reaction centers,

Acknowledgments This research was supported in large part by a Franco-Qu~bec Exchange grant held by C Lichtl6 and S Gibbs. We thank A Spilar and C Passaquet for their skillful technical assistance, and F Puel for preparation of the LH-antibody.

References 1 Andersson B, Anderson JM (1980) Lateral heterogeneity in the distribution of chlorophyll-protein complexes of the thylakoid membranes of spinach chloroplasts. Biochim Biophys Acta 593, 427-440 2 Armond PA, Staehelin LA, Arntzen CJ (1977) Spatial relationship of photosystem I, photosystem II, and the lightharvesting complex in chloroplast membranes. J Cell Biol 73,400-418 3 Berkaloff C, Duval JC, Hauswirth N, Rousseau B (1983) Freeze fracture study of thylakoids of Fucus serratus. J Phycol 19, 96-100 4 Bruce D, Biggins J, Steiner T, Thewalt M (1986) Excitation energy transfer in the cryptophytes. Fluorescence excitation spectra and picosecond time-resolved emission spectra of intact algae at 77 K. Photochem Photobiol 44, 519-525 5 Callahan FE, Wergin WP, Nelson N, Edelman M, Mattoo AK (1989) Distribution of thylakoid proteins between stromal and granal lamellae in Spirodela. Dual location of Photosystem lI components. Plant Physiol 91,629-635 6 Dodge JD (1969) The ultrastructure of Chroomonas mesostigmatica Butcher (Cryptophyceae). Arch Mikrobiol 69, 266-280 7 Dwarte D, Vesk M (1983) A freeze-fracture study of Cryptomonad thylakoids. Protoplasma 177, 130-141 8 Gantt E, Edwards MR, Provasoli L (1971) Chloroplast structure of the Cryptophyceae. Evidence for phycobiliproteins within intrathylakoidal spaces. J Cell Biol 48, 280-290 9 Gibbs SP (1970) The comparative ultrastructure of the algal chloroplast. Ann N Y Acad Sci 175, 454-473 l0 Guard-Friar D, Eisenberg BL, Edwards MR, MacColl R (1986) lmmunochemistry on cryptomonad biliproteins. Plant Physiol 80, 38-42 l l Hiller RS, Martin CD (1987) Multiple forms of type I phycoerythrin from a Chroomonas sp (Cryptophyceae) varying in subunit composition. Biochim Biophys Acta 923, 98-102 12 Hinshaw JE, Miller KR (1989) Localization of lightharvesting complex II to the occluded surfaces of photosynthetic membranes. J Cell Biol 109, 1725-1731 13 Ingram K, Hiller RG (1983) Isolation and characterization of a major chlorophyll a/c 2 light-harvesting protein from a Chroomonas species (Cryptophyceae). Biochim Biophys Acta 772, 310-319

194

C Lichtl~ et at

14 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lond) 227, 680-685 15 Lichtl~ C (1978) Etude exp6rimentale d'une Cryptophyc6e, analyse particuli6re de l'appareil photosynth6tique; comparaison avec quelques Rhodophyc6es. Th~se de Doctorat d'Etat, Paris 16 Lichtl6 C (1979) Effects of nitrogen deficiency and light of high intensity on Cryptomonas rufescens (Cryptophyceae). I. Cell and photosynthetic apparatus transformations and encystment. Protoplasma 101, 283-299 17 Lichtl6 C, Jupin H, Duval JC (1980) Energy transfers from photosystem I1 to photosystem I in Cryptomonas rufescens (Cryptophyceae). Biochim Biophys Acta 591, 104-112 18 Lichtl~C, Du~'al JC, Hauswirth N, Spilar A (1986) Freezefracture study of thylakoid organization of Cryptomonas rufescens (Cryptophyceae) according to illumination conditions. Photobiochem Photobiophys 11, 159-171 19 Lichtl~ C, Duval JC, Lemoine Y (1987) Comparative biochemical, functional and ultrastructural studies of photosystem particles from a Cryptophycea: Cryptomonas rufescens; isolation of an active phycoerythrin particle. Biochim Biophys Acta 894, 76-90 20 Ludwig M, Gibbs SP (1989) Localization of phycoerythrin at the lumenal surface of the thylakoid membrane in Rhodomonas lens. J Cell Biol 108, 875-884 21 Mullet JE, Arntzen CJ (1980) Simulation of grana stacking in a model membrane system. Mediation by a purified lightharvesting pigment-protein complex from chloroplasts. Biochim Biophys Acta 589, 100-117 22 Rhiel E, Kunz J, Wehrmeyer W (1989) Immunocytochemical localization of phycoerythrin 545 and of a chlorophyll a/c light-harvesting complex in Cryptomonas maculata (Cryptophyceae). Botanica Acta 102, 46-53 23 Rhiel E, M6rschel E, Wehrmeyer W (1985) Correlation of pigment deprivation and ultrastructural organization of thylakoid membranes in Cryptomonas maculata following nutrient deficiency. Protoplasma 129, 62-73 24 Rhiel E, M6rschel E, Wehrmeyer W (1987) Characterization and structural analysis of a chlorophyll a/c light harvesting complex and of photosystem I particles isolated from thylakoid membranes of Cryptomonas maculata (Cryptophyceae). Eur J Cell Biol 43, 82-92 25 Roth J (1982) The protein A-gold (pAg) technique a qualitative and quantitative approach for antigen localization on thin sections. In: Techniques in Immunocytochemistry, vol 1 (Bullock GR, Petruz P, eds) Academic Press, New York, 107-133 26 Setif P, Acker S, Lagoutte B, Duranton J (1980) Contri-

27 28

29 30

31 32

33

34

35 36

37

bution to the structural characterization of eucaryotic PS-I reaction centre. II. Characterization of a highly purified photoactive SDS-CPI complex. Photosynth Res 1, 17-27 Spear-Bernstein L, Miller KR (1985) Are the photosynthetic membranes of cryptophyte algae inside out ? Protoplasma 129, 1-9 Spear-Bernstein L, Miller KR (1987) Immunogold localization of the phycobiliprotein of a cryptophyte alga to the intrathylakoidal space. In: Progress in Photosynthesis Research, vol 2, sect 4 (Biggins J, ed) Martinus Nijhoff, Dordrecht, 309-312 Spear-Bernstein L, Miller KR (1989) Unique location of the phycobiliprotein light-harvesting pigment in the Cryptophyceae. J Phycol 25,412-419 Staehelin LA (1986) Chloroplast structure and supramolecular organization of photosynthetic membranes. In: Photosynthesis IlL Photosynthetic Membranes and Light-harvesting Systems (Staehelin LA, Arnzten C J, eds) Springer-Verlag, Berlin, 1-84 SweeneyBM (1981) Freeze-fractured chloroplast membranes of Gonyaulaxpolyedra (Pyrrophyta). J Phycol 17, 95-101 Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76, 4350-4354 Valion O, Wollman FA, Olive J (1985) Distribution of intrinsic and extrinsic subunits of the PS-1I protein complex between appressed and non-appressed regions of the thylakoid membrane : an immunocytochemical study. FEBS Lett 183, 245-250 Vallon O, Wollman FA, Olive J (1986) Lateral distribution of the main complexes of the photosynthetic apparatus in Chlamydomonas reinhardtii and in spinach: an immunocytochemical study using intact thylakoid membranes and a PS-II enriched membrane preparation. Photobiochem Photobiophys 12, 203-220 Wehrmeyer W (1970) Zur Feinstruktur der Chloroplasten einiger photoautotropher Cryptophyceen. Arch Mikrobiol 71, 367-383 Wehrmeyer W (1983) Phycobiliproteins and phycobiliprotein organization in the photosynthetic apparatus of Cyanobacteria, red algae and cryptophytes. In: Proteins and Nucleic Acids in Plant Systematics (Jensen U, Fairbrothers DE, eds) Springer-Verlag, Berlin, 143-167 Wehrmeyer W (1988) Structure of Cryptophyte photosynthetic membranes. In: Photosynthetic Light Harvesting Systems. Organization and Function (Scheer H, Schneider S, eds) W de Gruyter, Berlin, 35-47