Molecular architecture of the chloroplast membranes of Chlamydomonas reinhardi as revealed by high resolution electron microscopy

Printed i n Sweden Copyright © 1973 by Academic Press, Inc. All rights of reproduction in any form reserved

146

J. ULTRASTRUCTURE RESEARCH

44, 146-178 (1973)

Molecular Architecture of the Chloroplast Membranes of Chlamydomonasreinhardi as Revealed by High Resolution Electron Microscopy 1 FRANK KRETZER

Department of Biology, University of California, Los Angeles, California 90024 Received January 26, 1973, and in revised form March 5, 1973 The chloroplast membranes of Chlamydomonas reinhardi were prepared for thin section, high resolution electron microscopy with the specific goal of minimally denaturing the protein and with the least lipid solublization possible. Thus, membrane proteins were first cross-linked with a short exposure to 1% glutaraldehyde, briefly dehydrated in cold ethylene glycol, and infiltrated with Vestopal. After such a preparatory procedure, the partition membranes were 270 A thick, nearly three times the thickness of membranes preserved by conventional denaturing procedures (100 A). The 270 A-thick partition membranes showed a heterogeneous (10-100 A) globular substructure. Partition membrane thickness varied consistently from 100-270 A depending on the denaturing potential of the "fixative" and the hydrophobic character of the dehydrating agent. The 270 A-thick chloroplast membrane indicates that protein molecules must be arranged in a complex, condensed, three-dimensional arrangement with no continuous lipid bilayer as the basic component of the membrane. The work presented in this paper is an attempt to discern the molecular organization of the chloroplast membrane. The biochemical functions of these membranes are well defined--photosystem I with the reduction of NADP, photosystem II with photolysis of water, photophosphorylation, and an ordered electron transport chain (36, 37). These membrane bound phenomena are summarized in Table I. The quant u m efficiency (9) and the linear, ordered flow of electrons along a redox potential chain implies a high degree of molecular organization within the membrane. Such a complex molecular organization would maximize the statistical probability of orderly interaction between enzyme sequences by minimizing both randomness and distance between active sites. Many of the protein components have been isolated and characterized as to their molecular weights by various means, although m a n y of the heavier components may well be protein complexes (Table II). Yet the diversity 1 This investigation was supported by NSF Grant GB-7859 and USPHS 5 FOI-GM 49785-02.

147

CHLAMY CHLOROPLAST MEMBRANES

Table I. Speculative "Z-Scheme" for the Light Reactions of Photosynthesis (33) NADPH2<

(Vitamin K) plastoquinone

ferredoxin-NADP reductase j./~

ferredoxin

cytochrome b6

•

cytochrome c <~

cytochrome f

<

Mn chlorophyllb H20 .C1 ~-trap chlorophyll Ch1670 accessory pigments PHOTOSYSTEM II

plastocyanin

> 0 2

trap chlorophyll PToo Ch1680

PHOTOSYSTEM I

is obvious. Their approximate dimensions, assuming globular shapes, have been projected from their molecular weights, centrifugation behavior, or negative staining. Under these conditions, their dimensions would range from 25-100 A. The arrangement of these proteins in the membrane is unknown but it is generally considered that they are in the form of well-defined multienzyme complexes. Shadowcast isolated lamellae showed surface bumps 185 × 155 × t00 A in three types of distribution--random, linear, and crystalline (35). Freeze-etched lamellae displayed two distinct substructures (175 A and 110 A) in a pebbled background (5). Arntzen, Dilley, and Crane (2) associated photosystem I with the 110 A complex and photosystem II with the 175 A complex. There is no question that these surface bumps seen with the 40 A resolution of freeze-etched material reflect real substructure inherent in the membrane. Yet to perceive the molecular architecture which produces these bumps demands thin-sectioned material. The latter technique is the only technique that can provide the 5-10 A resolution needed to discern such molecular architecture. Thin-sectioned material implies that the tissue has been dehydrated and infiltrated with a plastic. If classical preparatory techniques are used (OsQ, KMnO4, or high

148

KRETZER

Table II. Molecular Weights of Proteins Located in Chloroplast Thylakoid Membranes and Their Estimated Globular Size

Protein

Molecular Weight

Globular Size

Cytochrome b Cytochrome c Plastocyanin Ferredoxin Structural protein Ferredoxin-NADP reductase NADP-cytochrome f reductase Cytochrome f Biotin carboxyl carrier protein Coupling factor Ca2+-dependent ATPase Carboxydismutase

12 000 12 000 13 000 15 000 25 000 42 000 45 000 50 000 100 000 250 000 350 000 550 000

25 A a 25 t~a 25 Aa 25 A a 40 A ~ 50 A b 50 A b 55 A b 70 •b 90 Ab 100 Ae 110 A e

Reference

(17) (17) (16)

(17) (7)

(49) (62)

(33) (6)

(42) (20) (20)

a Projected from Dickerson's (8) X-ray crystallographic data on cytochrome c. b Centrifugation behavior. e Negative staining.

concentrations of glutaraldehyde fixation, and acetone or alcohol dehydration), the resultant membranes are denatured, extracted remnants of the previous in vivo structure (24, 27, 55, 56) and a 10 A analysis of the rearranged random-coiled protein is useless if molecular architecture is to be considered. Anticipating a complex chloroplast membrane structure from Tables I and II, steps were taken in the tissue preparation to minimize protein conformational changes according to the procedure developed by Sj 6strand and Barajas (55, 56). The basic principles involved were as follows: (a) stabilization of the native conformation of globular proteins by intermolecular crosslinking via a short exposure to a low concentration of glutaraldehyde as cross-linking agent; (b) short dehydration in ethylene glycol as an agent which affects the native conformation of proteins less drastically than acetone or any other organic solvent; (c) embedding in a polar plastic, Vestopal. The theoretical basis for this procedure has been discussed by Sj6strand and Barajas (55, 56). In this study, Chlamydomonas reinhardi was chosen as the source of chloroplast membrane because of the small size of the cells, the peripheral cup-shaped chloroplast, the ease with which they are cultured, and the availability of photosynthetic mutants which affect single protein components along the photosynthetic electron transport chain (14-18, 28, 30). The Sj~Sstrand-Barajas technique was modified for dispersed cells and the cross-linking and dehydration times could be reduced to the physical minima of diffusion times through a single cell.

CHLAMY CHLOROPLAST MEMBRANES

149

METHODS

Culturing of cells Cultures of Chlamydomonas reinhardi wild-type strain 137C (mt +) or the mutant strain ac-206 (18) were grown in sterile, autoclaved liquid medium in l-liter Bellco spinner flasks at 24°C. The growth medium (29) contained the following: 50 ml/liter Beijerinck's solution (1 g NH~C1, 0.4 g K2HPO4, 0.4 g MgSO~.7H20, 0.2 g CaCI~.2H20, 1 liter H20), 50 ml/ liter phosphate buffer (23.22 g K~HPO~, 18.14 g KH2PO4, 1 liter H~O), 10 ml/liter NH~C1 solution (45 g/liter H~O), 1 ml/liter trace element solution (25 g EDTA, 11 g ZnSO~-7H20, 5.7 g H3BOa, 2.53 g MnCI~'4H~O, 2.45 g FeSO4' 7H~O, 0.80 g CoCI~.6H=O, 0.78 g CuSO45H~O, and 0.55 g (NH~)6MoTO~'4H~O), and 2 g/liter sodium acetate. Cultures were incubated for 36-48 hours while stirred on a magnetic stirrer, bubbled with air filtered through layers of cotton, and illuminated continuously with cool-white fluorescent lights. While still in the log growth phase (approximately 5 × 10" cells per ml), the cells were harvested by centrifugation for 5 minutes at 5 000 rpm in a SS-34 Servall rotor at 0°C. Twenty 50-ml samples were obtained from each l-liter culture.

Agar encasements vs free cells The resultant pellet of cells could be handled in two ways: 1. The decanted 50 ml centrifuge tube was inverted on filter paper so that the pellet was drained of excess moisture. Then the pellet was scooped up with a weighing spatula, spread over a 1.5 cm area of a slightly warmed slide (30°C), and mixed with a drop of 3 % agar made with the supernatant medium which had been cooled in advance from 100°C to 30°C over a period of 90 minutes. The best infiltration and sectioning resulted when the cell concentration was one pellet per drop of agar. The cooled solidified agar slab was cut into about 50-/~m sections with a razor blade and treated as pieces of "tissue" for fixation, buffer washes, dehydration, and infiltration. 2. Approximately one-fifth of the original pellet was mixed with the "fixation" solution in a 1-ml plastic centrifuge tube of a Spinco table Model 152 Microfuge. After the desired fixation time (which could be greatly reduced because the limiting factor here was diffusion through a single cell as opposed to diffusion through an agar block), the cells were centrifuged for 30 seconds. Two cuts with a razor blade were made in the centrifuge tube above and below the pellet forming a cylinder of plastic around a core of fixed cells. The plastic cylinder was held with forceps and cells were collected on the tip of a wooden tooth pick pushed through the central core. The isolated clump of cells was then resuspended in 1 ml of the wash buffer. This process of centrifugation and collection was repeated as the dispersed cells were transferred from buffer wash to dehydration solution to infiltrating plastic, As long as the solution of the liquid growth medium was used as the buffer for the "fixative" and for the washes, no cell explosion was observed in this dispersed cell technique.

Buffers for the fixative and the buffer wash Various buffers were tried (Veronal-acetate, collidine (38), Maunsbach phosphate (54), cacodylate, and barbital), but the buffer of choice was the supernatant medium from the liquid growth culture. Fixed dispersed cells were given a 5 minute buffered 0°C wash, while fixed agar blocks were washed for 30 minutes.

150

KRETZER

"Fixation" procedures All fixations were performed at 0°C. The 1% OsO4 solution was made 24 hours before use. The 1% and 6 % glutaraldehyde solutions were made minutes before use. The purified glutaraldehyde 1 gave more consistent results than the heterogeneous bulk commerical glutaraldehyde, which before use was filtered over activated charcoal. The shortest fixation time which preserved the cells in the agar blocks was 30 minutes, while dispersed cells could be optimally preserved with 1 minute "fixation."

Dehydration procedures Neither dispersed cells nor agar blocks required nor showed any improved quality with graded, slow dehydration sequences. Dispersed cells were dehydrated in 1 minute with 100% 0°C acetone dried over CuSO4 or by 1 minute in 0°C ethylene glycol. The shortest acetone dehydration of agar blocks which yielded well infiltrated cells was 30 minutes in 100% 0°C acetone. Ethylene glycol dehydration of agar blocks was performed in the following way. Agar blocks were placed on the surface of 8 ml of ethylene glycol at 0°C in 10-ml vials. After the pieces settled to the bottom (10-30 minutes), they were picked up with a toothpick, drained with strips of filter paper, and transferred to 100% Vestopal in 10-ml vials.

Infiltration procedures Either Vestopal W (45) or

the low viscosity Spurr Epoxy resin (58) was used as the infiltrating plastic. In both cases, polymerization occurred in a 45°C oven for 1 week. Such polymerization was found to be superior to the 8-hour 70°C cure as proposed by Spurr. The lower temperature minimized protein denaturation, and the longer time resulted in a better cure of the plastic as determined by ribbon length and the appearance of the trimmed sides of the block. The infiltrated agar blocks or dispersed cells were finally embedded in gelatinous capsules which had previously dried for 1 week at 100°C. Dispersed cells, dehydrated with either acetone or ethylene glycol for as short a period as 1 minute, were infiltrated with Vestopal in the following way: The dehydrated pellet was resuspended in a 1-ml plastic centrifuge tube for the Spinco 152 Microfuge in 1 ml of Vestopal at 24°C (with 1% initiator and 1% activator) for 5 minutes. The infltrated ceils were then centrifuged for 30 seconds at 24°C and the isolated pellet was resuspended in Vestopal in gelatinous capsules. Five to six such pellets were pooled in one such capsule. Thus, the entire capsule contained an even suspension of cells. If desired, ethylene glycol-dehydrated single cells could be transferred to 10-ml vials 3/4 filled with Vestopal (with no initiator and no activator.) Such closed vials were placed on a 360 ° rotating wheel (one revolution every 4 minutes), and constantly tumbled for 5-14 days at 24°C to determine the effect of Vestopal on chlorophyll extraction. (Such long periods were not due to problems of infiltration, since this occurred optimally in as short a time as 5 minutes.) On the last day of these prolonged Vestopal infiltrations of dispersed cells, 1% initiator was added to the 10-ml vials, the vials closed up, returned to the rotating wheel, and tumbled for 2 hours at 24°C. Then 1% activator was added, and the vials were again tumbled for 22 hours at 24°C. The cells were centrifuged from the Vestopal by 10 1 Purchased from Ladd Research Industries Inc. in sealed 2-ml vials.

CHLAMY CH LOROPLAST MEMBRANES

151

minutes at 10 000 rpm in a SS-34 Servall rotor at 10°C. The pellet was resuspended in capsules and polymerized. Ethylene glycol-dehydrated dispersed cells were infiltrated with Spurt Epoxy resin (58). For this, an equal volume of the 0°C plastic (10 mg ERL-4206, 4 mg DER-736, 26 mg NSA, 0.4 mg s-l) was added to the suspension of dehydrated cells still in the 0°C ethylene glycol solution. After 3 minutes of centrifugation at 10 000 rpm in a Servall SS-34 rotor at 0°C, the pellet was resuspended in fresh Spurr plastic and centrifuged for another 3 minutes. The pellet of cells was then mixed with more Spurr plastic in dried gelatinous capsules and polymerized. Dried capsules were essential, otherwise the plastic was very soft. Spurr embedded specimens did not section in long ribbons, but the sections were thin. The Epoxy resin is miscible with ethylene glycol despite the admonition of Spurr (58). Vestopal infiltration of ethylene glycol-dehydrated agar blocks proved to be a problem~ It was determined that if the dehydrated agar blocks were tumbled for 4 days at 24°C in Vestopal without initiator and activator no polymerization of Vestopal would occur increasing the chances of Vestopal mixing with the ethylene glycol. After 4 days, 1% initiator was added to the 10-ml vials, and then the vials were tumbled for an additional 2 hours at 24°C, 1% activator was added, and the vials were tumbled for 22 hours at 24°C. The agar pieces were transferred to gelatinous capsules and polymerized. One out of every 8 capsules was successfully infiltrated. This emphasizes the difficulty of Vestopal infiltration of "large pieces" of ethylene glycol-dehydrated tissue and shows the desirability of the dispersed cell system for attaining short fixation, dehydration, and infiltration times.

Electron microscopy Blocks were trimmed by hand to a rooftop-shaped tip 0.1 mm long with smooth sloping sides subtending a 3 mm square base. Such uniformly cut blocks were sectioned with an LKB 4 800 Ultrotome equipped with glass knives using a boat made with silver photographic tape containing filtered triple-distilled water. Sections were picked up on single-hole grids covered with 0.3 % Formvar films or Triafol nets (10). The sections were section-stained for 1 hour in a 60°C oven in a saturated solution of uranyl acetate (8 g/100 ml triple-distilled water, made 24 hours prior to use, stored at 24°C permit to saturation, and filtered 3 times through U F glass filters prior to use). The stained grids were then washed by 15 dips in each of five 10-ml vials filled with filtered triple-distilled water at 60°C. The grids were then very lightly carbon-coated and examined on a Siemens Elmiskop I at 80 kV with 200/~m molybdenum condenser apertures and 50 ~m molybdenum objective apertures. The sections were examined at 40 000 and 80 000 times electron optical magnification.

Measurements To eliminate bias when determining chloroplast membrane thickness, a Plexiglas sheet with random scratched non parallel lines was randomly placed over glossy prints of negatives taken initially at 40 000 or 80 000 times electron optical magnification and enlarged 3 times photographically. Where two scratched lines intersected a membrane cut in cross section, a measurement was made. After 100 such measurements, the mean value was determined. Twenty-five additional measurements were made. The mean was determined for these 125 measurements, and the process continued until the new mean was identical to the previous one. Each experiment to determine membrane thickness was repeated three times to check reproducibility. Data was collected from a minimum of 30 different cells in each case.

152

KRETZER

To reduce bias when analyzing the particulate substructure, measurements were made on glossy prints of obliquely cut membranes from negatives taken initially at 40 000 and enlarged photographically ten times from the close-to-focus picture from a through focus sequence. The distance between the middle of the dark limiting line to the middle of the opposite dark limiting line along the longest diameter of the particle was defined as the width of such particles. Randomly chosen particles were assured by using the plexiglass sheet method described above. Three hundred measurements of particle diameters (30 particles from each of ten different cells in the thinnest section of a ribbon) were made and grouped in 10 A intervals as the percentage of particles counted rounded off to the nearest 5 %. This was called the particle histogram. Each experiment to determine particle diameters was repeated three times to check reproducibility. The thickness of the section from which the pictures were taken would definitely influence particle size. The thicker the section, the larger the particles would be due to superposition effects. To reduce this factor, the same sized pyramids were constantly cut, ribbons were the result of the same low thermal expansion so that one section was produced per 4-5 cycles of the cutting arm, and pictures were taken only from the apparent thinnest section along any given ribbon as viewed at low magnification in the electron microscope. Thus the sections from which pictures were taken for measurements represented the thinnest possible sections. The parameter of section thickness was assumed to be constant enough so that particle size differences were not due to significant variations in section thickness. [This was verified by the fact that 300 particles counted from 10 different cells (from 10 different experiments) where each of the 10 cells was in the thinnest section of the 10 respective ribbons (Table IV) produced the same histogram as 300 particles counted from 10 different cells, where each of the cells was in the same single apparently thinnest section of one ribbon from one experiment (Table III).] There were no significant fluctuations from cell to cell, emphasizing the homogeneity of this dispersed cell system.

Triafol nets Due to the inherent low contrast of the glutaraldehyde-ethylene glycol treated tissue, Triafol nets were used to support thin sections over one-hole, slot copper grids. SjSstrand in 1956 (51) developed a method to make Formvar nets in an attempt to improve contrast of thin sections prior to the currently used section staining techniques. The thinnest stable supporting Formvar film is about 200 ~ thick. When compared to the 100 .~-thick sections required for high resolution electron microscopy, the 200 A-thick film contributes greatly to random scattering of electrons and thus reduces contrast by increasing the background noise. Therefore it became necessary to suspend these thin sections over small 2 #m holes in the supporting nets. Such thin sections did not rupture when they were extended over sufficiently small holes. Triafol nets were found to be more stable and to afford a greater control of hole size than Formvar nets. The method of Fukami and Adach ~, (10) was modified so that high quality uniform, stable nets could be used for daily sectioning. The following procedure was routinely performed using supplies bought from Maeoma Inc. Tokyo, Japan, to produce nets with a 2-/~m hole size. The timing was very critical and must be empirically adjusted if room conditions changed or other parameters were varied. Glass slides were scrubbed with fine steel wool and polished with Ross Optical lens tissue. The cleaned slides were soaked for 2 days in 4 changes of 0.03 % Softex (adjusted from the percentage of the bought stock solution with triple-distilled water) and then were

CHLAMY CHLOROPLAST MEMBRANES

153

washed by 10 dips in each of 5 containers of triple-distilled water. These water-repellent treated slides dried overnight in a desiccator with anhydrous CaSO4 H a m m o n d Drierite. The Triafol was dissolved in Mallinckrodt ethyl acetate to form a 0.5 To (w/v) solution, was filtered 3 times through U F glass filters, and was refrigerated. The dried slides were put into a freezer on a brass plate for 5 seconds, and then held 9 inches in front of a DeVilbriss No. 145 vaporizer filled with tap water for 5 seconds. The slide was plunged down into the cold Triafol solution for 11 seconds and drained in a refrigerator for 10 minutes. The resultant nets were examined under a phase contrast microscope. If the net was acceptable, the slide was soaked for 4 hours in 0.5 To Pelex (adjusted from the percentage of the bought stock solution with triple-distilled water), the sides were scratched with a razor blade, and the nets floated off onto a 5-inch diameter glass container filled with triple-distilled water. The net was covered with one-hole, slot copper grids which had previously been nitric acid etched and dipped in 0.1% Triafol (w/v) in ethyl acetate. The nets were then collected on Parafilm and stored in covered petri dishes. Four parameters can be manipulated in determining net size: (a) time on brass plate in freezer, (b) time in front of vaporizor, (c) time in cold Triafol, and (d) time drained in refrigerator. However, stability of the nets in the electron beam depended solely on the meticulous cleanliness achieved during their preparation. This cleanliness has three ramifications: (a) All solutions were made with triple-distilled water and filtered through glass filters cleaned with concentrated sulfuric acid. (b) The ethyl acetate was filtered through U F glass filters cleaned with concentrated sulfuric acid. (c) Glass slides were cleaned with steel wool and polished with lens paper. Any short cuts resulted in film rupture or drift. The water-repellent treatment was greatly extended since the original time was too short for proper slide-wetting. Ribbons floating on the surface of the triple-distilled water in the boat after sectioning were difficult to pick up from underneath when Triafol nets were used. A modification of the Galey-Nilsson (11) method was thus routinely employed. The ribbon on the surface of the water was caught from above in the hole of a clean, filmless grid held by a pair of forceps. This grid's hole was aligned with that of the net-filmed grid, released from the forceps, and placed on top of the net-filmed grid. A second pair of forceps holding the net-filmed grid prevented the two grids from large area contact. As the water dried, the ribbon flattened out without wrinkles onto the net film and the empty grid was tapped off.

RESULTS Technically it was possible to reduce b o t h cross-linking time with 1% glutarald e h y d e a n d ethylene glycol d e h y d r a t i o n to 1 m i n u t e each when dispersed cells were used. N e i t h e r the 1-minute d e h y d r a t i o n or the 5-minute V e s t o p a l infiltration of these d i s p e r s e d cells e x t r a c t e d any visible chlorophyll. The short infiltration p e r i o d created no p r o b l e m s in s u b s e q u e n t sectioning. I n 1-minute 1% g l u t a r a l d e h y d e cross-linked, 1-minute ethylene glycol d e h y d r a t e d , 5-minute V e s t o p a l infiltrated wild-type cells (25), the cross-sectioned c h l o r o p l a s t m e m b r a n e s were u n i f o r m l y 270 A thick. T h e adjacent, t h i c k c h l o r o p l a s t m e m b r a n e s were closely p a c k e d , a n d the m e m b r a n e s t e r m i n a t e d bluntly at the ends of the m e m -

154

KRETZER

T a b l e III. Particle Distribution in Ten Ceils from One Experiment ~ Cells A

B C D E F G H I J Total Percent

10-20 A 2 0 - 3 0 A 30-40 A 40-50 A 50-60 A 60-70 A 70-80/~ 80-90 A 90-100 A 2

•

2 1 I 3 1 10 3.32

•

- -

7 3

10 10

1

2 4 5 4 2 6 6

4 2 5 3 3 6 l l 3 23 9.3

40 13.3

2 --

-2

4 3

4 7

10

3

11 13 12 6 8 8 12

-1 1 1 3 -3

100 33.3

14 4.7

1 1

--

1

10

1

4 -2 --5 --

-2 -5 ----

8 5 4 6 11 8 5

-1 2 2 2 2 --

13 4.3

15 5.0

68 22.7

12 4.0

a 300 particles measured from 10 different cells in.the thinnest section of one ribbon from one experiment. The wild-type cells were cross-linked for 1 minute with 1% glutaraldehyde, dehydrated 1 minute in 0°C ethylene glycol, and infiltrated for 5 minutes in Vestopal.

b r a n e stacks (Fig. 1). T h e g e n e r a l i m p r e s s i o n of these c r o s s - s e c t i o n e d m e m b r a n e s w a s t h a t t h e edges wei:e o p a q u e a n d t h e r a t h e r lucid g i r t h of t h e m e m b r a n e was f a i n t l y d e l i n e a t e d i n t o areas of v a r i o u s d i m e n s i o n s (Fig. 2). O n o b l i q u e l y s e c t i o n e d m e m b r a n e s , it was a p p a r e n t t h a t the t h i c k m e m b r a n e m a s s was c o m p o s e d of i n d i v i d u a l n o n u n i f o r m p a r t i c l e s w h o s e n o n s t a i n i n g c e n t r a l a r e a was d e m a r c a t e d b y a faint, o p a q u e 10 A b o r d e r (Figs. 3 a n d 4). A t h i g h e r m a g n i f i c a t i o n of s u c h o b l i q u e l y (Fig. 5) a n d t a n g e n t i a l l y (Fig. 6) c u t c h l o r o p l a s t m e m b r a n e s , t h e i n d i v i d u a l h e t e r o g e n e o u s p a r ticles w e r e o b s e r v e d

and

measured. In Graph

1, 300 p a r t i c l e s f r o m 10 d i f f e r e n t

Chlamydomonas reinhardi w i l d - t y p e c e l l s in the s a m e t h i n n e s t section of o n e r i b b o n f r o m o n e e x p e r i m e n t ( T a b l e I I I ) w e r e m e a s u r e d a n d r e c o r d e d in 10 A i n t e r v a l s a s t h e p e r c e n t a g e of p a r t i c l e s c o u n t e d r o u n d e d off to t h e n e a r e s t five p e r c e n t . T h e p a r ticles r a n g e d f r o m 10 t o 100 A in d i a m e t e r ( T a b l e s I I I a n d IV). T h e h i s t o g r a m h a d F i e 1. Chloroplast membranes in Chlamydomonas reinhardi, cross-linked for 1 minute with 1% glutaraldehyde, dehydrated for 1 minute in 0°C ethylene glycol, and infiltrated for 5 minutes in Vestopal. These 270 A-thick membranes, seen here in cross section, are closely packed displaying opaque edges and lucid girth. The adjacent membranes terminate abruptly at certain points (arrows), x 180 000. Fro. 2. Chloroplast membranes in Chlamydomonas reinhardi, cross-linked for 1 minute with 1% glutaraldehyde, dehydrated for I minute in 0°C ethylene glycol, and infiltrated for 5 minutes in Vestopal The 270 Arthick membranes, seen here in cross section, show a particulate substructure (arrow). × 224 000. Fro. 3. Chloroplast imembranes in Chlamydomonas reinhardi, cross-linked for 1 minute with 1% glutaraldehyde, dehydrated for 1 minute in 0°C ethylene glycol, and infiltrated for 5 minutes in Vestopal. These membranes, oriented obliquely in relation to the plan e of the section, show a very distinct particulate substructure (arrows). × 224 000.

!

i~ iil,

158

KRETZER

Particle Distribution in Ten Cells from Ten Different Repeats of the Same Experiment ~

T a b l e IV.

Cells

10-20 A 20-30 ~ 30-40 ~ 40-50 ~ 50-60 ~ 60-70 ~ 70-80 ~ 80-90 ,~ 90-100 A

A B C D E F G H I J

1 1 1

2 1 1 2

2 2 2 5 1 6 3 3 3 4

7 2 5 6 5 4 5 6 3 1

12 4

31 10.3

44 14.7

3

Total Percent

9 10 5 10 10 5 11 15 12 10

---2 --4 -1 4

1 3 1 -3 1 --3 --

2 1 3 --2 --2 4

7 8 13 7 6 12 5 4 4 4

97 32.3

11 3.7

12 4

14 4.7

70 23.4

1 3 --2 --1 1 1 9 3

a 300 particles measured from 10 different cells. Each cell was in the thinnest section of the respective ribbon of 10 different repeats of the same preparatory technique. Wild-type cells were crosslinked for 1 minute with 1% glutaraldehyde, dehydrated 1 minute in 0°C ethylene glycol, and infiltrated for 5 minutes in Vestopal.

b i m o d a l p e a k s a t t h e 40 a n d 80 A i n t e r v a l s . S i x t y p e r c e n t of t h e p a r t i c l e s w e r e s m a l l e r t h a n 50 A . In 1-minute 1% glutaraldehyde cross-linked, 1-minute ethylene glycol dehydrated, 5-minute Vestopal infiltrated mutant

(ac-206) cells, t h e c h l o r o p l a s t m e m b r a n e s w e r e

2 7 0 A t h i c k , c l o s e l y p a c k e d , a n d t e r m i n a t e d a b r u p t l y a t t h e e n d s of t h e m e m b r a n e stacks. Again, the cross-sectioned membranes had opaque edges and the lucid girth was

f a i n t l y d e l i n e a t e d i n t o a r e a s of v a r i o u s d i m e n s i o n s . 300 p a r t i c l e s (30 p a r t i c l e s

f r o m e a c h of 10 d i f f e r e n t cells i n t h e s a m e t h i n n e s t s e c t i o n of a r i b b o n ) of o b l i q u e l y and tangentially cut

ac-206 c h l o r o p l a s t m e m b r a n e s w e r e m e a s u r e d a n d r e c o r d e d i n

10 A i n t e r v a l s a s p e r c e n t a g e of p a r t i c l e s c o u n t e d r o u n d e d

off t o t h e n e a r e s t 5 %.

The results were reproducible and constant in three separate experiments. As with FIG. 4. Chloroplast membranes in Chlamydomonas reinhardi, cross-linked for 1 minute with 1% glutaraldehyde, dehydrated for 1 minute in 0°C ethylene glycol, and infiltrated for 5 minutes in Vestopal. These membranes, oriented obliquely in relation to the plane of the section, show a very distinct particulate substructure (arrows). x 224 000. FIG. 5. Chloroplast membranes in Chlamydomonas reinhardi, cross-linked for 1 minute with 1% glutaraldehyde, dehydrated for 1 minute in 0°C ethylene glycol, and infiltrated for 5 minutes in Vestopal. These membranes, oriented obliquely in relation to the plane of the section, show at this high magnification the heterogeneous particulate substructure as nonstaining central areas demarcated by faint opaque 10 A borders (arrows). x 1 200 000. FIo. 6. Tangential view of chloroplast membranes in Chlamydomonas reinhardi, cross-linked for 1 minute with 1% glutaraldehyde, dehydrated for 1 minute in 0°C ethylene glycol, and infiltrated for 5 minutes in Vestopal. The particulate substructure is obvious (arrows). x 1 200 000.

11 -

731827

J. Ultrastructure Research

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GRAPHS 1--3. Distribution of particle diameters as measured in obliquely oriented chloroplast parti-

tion membranes i n 10 A intervals, rounded off to-the nearest 5 %.

FIG. 7. Chloroplast membranes in Chlamydomonas reinhardi, cross-linked for 1 minute with 6 % glutaraldehyde, dehydrated for 1 minute in 0°C ethylene glycol, and infiltrated for 5 minutes in Vestopal. The closely packed membranes are 150 A thick and at points terminate bluntly as the end of the membrane mass (arrows). A faintly stained line appears down the center of some of the membranes. × 160 0 0 0 . FIG. 8. Chloroplast membranes in Chlamydomonas reinhardi, cross-linked for 1 minute with 1% glutaraldehyde, dehydrated for 1 minute in 0°C ethylene glycol, and infiRrated for 6 minutes in 0°C Spurr Epoxy resin. This cross section shows that the chloroplast membranes are 220 A thick, closely opposed, and terminate abruptly at the apical e n d of the cup,shaped chloroplast as an uneven package of membrane material (arrows). A faintly stained line appears d o w n the center of the membranes. × 120 000.

164

KRETZER

the wild type, the same nonuniform particle range existed (10-100 ~ ) and the bimodal peaks of the resultant histogram occurred morphologically at the same locations (Graph 2). However, in the ac-206 photosynthetic mutant, 55 % of the particles were smaller than 50 A in contradistinction to 60 % for the wild type. This 5 % decrease in smaller than 50 A particles appeared to be due to changes in the 20-30 and 30-40 A intervals. The effects of both cross-linking and dehydrating agents on membrane structure were analyzed. If both the one-minute ethylene glycol dehydration and 5-minute Vestopal infiltration were held constant, and the 1% glutaraldehyde cross-linking time was increased to 5, 10, and 15 minutes, respectively, then the chloroplast membrane dimension decreased to 250 A in the first two instances and to 210 A in the latter. With 15-minute cross-linking, a faint dense line was perceived down the center of the 210 A-thiCk chloroplast membrane. In all three cases, the thick adjacent membranes were closely apposed. In the 5- and 10-minute cross-linked membranes, their girth was still subdivided into particles of various sizes. On oblique sections of the 10-minute cross-linked wild-type chloroplast membranes, a detailed particle analysis was carried out (Graph 3). 300 particles (30 particles from each of 10 different ceils in the same thinnest section of one ribbon) were measured and recorded in 10 A intervals as percentage of particles counted rounded off to the nearest 5 %. When the resultant particle size histogram from cells which had been exposed to 10-minute cross-linking was compared with that of the 1minute cross-linked wild-type strain, the particle size range appeared to have shifted from 10-100 A for the latter to 20-120 A for the former. The bimodal character of the~ l~istogram seemed to be preserved, but t h e peaks presumably had shifted to 60 and 90 A. After 10-minute cross-linking, only 15 % of the particles measured smaller than 50 A.: Thus, the particles appeared to enlarge (20 120 A) as the membrane thinned t o 250 A. Again the data were constant and reproducible in three separate experiments. If cells were exposed to the same 1-minute ethylene glycol dehydration and 5minute Vestopal infiltration as above, but the 1-minute cross-linking was with 6 % glutaraldehyde, the closely packed chloroplast membranes shrank to 150 A (Fig. 7). The mitochondrial inner membranes from the same 30 cells were often 100 A in

FIG. 9. Chloroplast membranes in Chlamydomonas reinhardi, cross-linked for 1 minute with 1% glutaraldehyde, dehydrated for 1 minute in 0°C absolute acetone, and infiltrated for 5 minutes in Vestopal. The membranes are 100 A thick. A Golgi apparatus is visible in the lower right-hand corner of the picture, x 120 000. FiG. 10. Chloroplast membranes in Chlamydomonas reinhardi, merely dehydrated for 1 minute in 0°C ethylene glycol and infiltrated for 5 minutes in Vestopal. The membranes are 100 N thick. x 120 000.

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contradistinction to the 300 A thickness in the 1% glutaraldehyde cross-linked material. If both the 1-minute cross-linking and 5-minute Vestopal infiltration were held constant, but the dispersed Cells were exposed to 30 minutes a n d 60 minutes of ethylene glycol dehydration, then the closely packed Chloroplast membranes were 150 and 100 A thick, respectively. All the chlorophyll was extracted during these prolonged ethylene glycol dehydrations. However, with a brief 1 minute glutaraldehyde crosslinking as a stabilizer of membrane molecular architecture, it took 60 minutes of adverse exposureto 100 % ethylene glycol for the membrane to be reduced to 100 A. On the other hand, if fresh cells were merely dehydrated by 1-minute exposure to ethylene glycol and then infiltrated for 5 minutes in Vestopal, the membranes were 100 A thick (Fig. 10). This paralleled Pease's (39) "inert dehydration" except that the ethylene glycol exposure was one-sixth as long and there was no Cellosolve intermediate. The cells did not explode, cell morphology was basically preserveG but the chloroplast membranes were nearly one-third the size of chloroplast membranes exposed to the same ethylene glycol dehydration time but previously stabilized by 1minute cross-linking. Five to 14 days of 24°C infiltration with Vestopal of 1-minute cross-linked, 1minute ethylene glycol-dehydrated dispersed ceils did not extract the chlorophyll, perhaps reflecting the polar nature of Vestopal. If such cells were infiltrated with a m o r e nonpolar embedding medium such as the low viscosity Spurt Epoxy resin (58) for 6 minutes at 0°C, all the chlorophyll was extracted. After such chlorophyll extraction, the chloroplast membranes were 220 A thick closely apposed, terminating abruptly at the apical end of the chloroplast as an uneven package of m e m b r a n e material (Fig. 8). Chlorophyll extraction implies that the Spurr Epoxy resin interferes with hydrophobic interactions in the membrane and that it is less polar than Vestopal. Thus, as the parameters of the original Sj6strand-Barajas scheme were varied (length and concentration of glutaraldehyde cross-linking, extent of ethylene glycol FIG. 11. Chloroplast membranes in Chlamydomonas reinhardi, fixed for 1 minute in 1% OsO4, dehydrated for 1 minute in 0°C absolute acetone, and infiltrated for 5 minutes in Vestopal. The intact cell wall (CW)and plasma membrane (PM)are visible. The chloroplast is composed of long parallel disks, thylakoids, which appear to be stacked and fused together. At the extreme tips of these disks, the 50 ~_-thick membrane loops around and fuses with the adjacent membrane of the opposing disk to form 100 A partition membranes. The arrow shows isolated points where there is a separation of the partition membrane into its two fusion components. The contained phase between the partition membranes is called the loculus (L), and the area outside these disks is called the stroma (5). The single 50 A membrane facing the stroma is called the end membrane (EM). x 120 000. FIG. 12. Chloroplast membranes in Chlamydomonas reinhardi, fixed for 1 minute in 1% OsO4, dehydrated for 1 minute in 0°C absolute acetone, and infiltrated for 5 minutes in Vestopal. In certain areas (arrows), the partition membranes are 250 A thick and are composed of two opaque outer lines separated by a clear area transversed by faint opaque septa, x 200 000.

12 -- 731827

J. Ultrastructure Research

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KRETZER

dehydration, and the apparent polar Vestopal infiltration contrasted with the apparent nonpolar Spurr Epoxy resin) concomitant membrane changes were observed. It seemed justifiable to determine what the conventional techniques would produce morphologically when used for equally short periods of time on dispersed cells. Oneminute 1% OsOa fixation, followed by 1-minute absolute acetone dehydration, and 5-minute Vestopal infiltration produced nonexploded cells in which the chloroplast was composed of long parallel disks (thylakoids) which appeared to be stacked and fused together (Fig. 11). At the extreme tips of these disks, the 50 A-thick structureless membrane looped around and fused with the adjacent membrane of the apposing disk to form partition membranes which were 100 • thick. The contained phase between partition membranes is called the loculus, in contradistinction to the area outside these disks, the stroma (21, 47). The single membrane facing the stroma is called the end membrane and was 50/k thick. The contrast was high and the result was the classic picture of algal chloroplast membranes (no differentiation into isolated granal stacks) (12). There was little gain in information as exposure to both 1% OsO4 and acetone dehydration were reduced from 1 hour, to 0.5 hour, to 1 minute. In fact, at the 1 hour and 0.5 hour periods, the dispersed cells vs. the agar blocks of cells were identical. However, two morphological situations were peculiar to the 1-minute material: (a) At isolated points it was obvious that there was a separation of the partition membrane into its two fusion components (Fig. 11). (b) At very rare points, the partition membranes were 250 A thick. Such thick membranes were composed of two opaque outer lines separated by a clear area transversed by faint, opaque septa (Fig. 12). If the 1-minute OsO4 was replaced with 1-minute 1% glutaraldehyde, followed by 1-minute acetone dehydration, the same 100 • image was seen, but only in reversed contrast (Fig. 9). These results were summarized in Table V. DISCUSSION With minimum cross-linking and exposure to ethylene glycol, the chloroplast membranes are closely packed, 270 A-thick structures, whose girth is composed of heterogeneous areas delineated by faint 10 A border demarcations. Each of these three morphological features (width, packing, and substructure) respond to increased cross-linking and dehydration times. With increased cross-linking to 15 minutes, the membrane thins to 210 A and the individual particles morphologically enlarge (Graph 2). With increased exposure to ethylene glycol, the membrane thickness is reduced to a structureless 100 A membrane. Brief exposure to OsO4 and acetone produces great variations in width, packing, and substructure. The membrane thins

169

CHLAMY CHLOROPLASTMEMBRANES Table V. Data S u m m a r y

Preparatory Technique 1 minute 1% glu, 1 minute ethylene glycol 5 minutes 1% glu, 1 minute ethylene glycol 10 minutes 1% glu, 1 minute ethylene glycol 15 minutes 1% glu, 1 minute ethylene glycol 1 minute 6 % glu, 1 minute ethylene glycol 1 minute 1% glu, 30 minutes ethylene glycol 1 minute 1% glu, 60 minutes ethylene glycol no cross-linking, 1 minute ethylene glycol 1 minute 1% glu, 1 minute ethylene glycol, 6 minutes O°C Spurr

1 minute 1% OsO~, 1 minute acetone

1 minute 1% glu, 1 minute acetone

Membrane Thickness Figure (A) No. 270~ 25O 250 210 150 150 100 100

1-6

7 10

220 8 100 11-12 (rare isolated 250 A segments) 100 9

to 100 A, is structureless, and separates into what appears to be a system of disks with the emergence of thinner end membranes and thicker partition membranes. There is no doubt that the conventional technique produces a denatured, extracted remnant of the previous in vivo structure. The studies of Lenard and Singer (27) showed that 2 % OsO 4 destroyed 63 % of the c~-helix content of intact red blood cell membrane proteins as monitored by circular dichroism in the UV range. With the rigid e-helix content of the membrane proteins randomized, reorganization of the protein material results in new hydrophilic surfaces which separate into the disklike appearance of chloroplast lamellae which are characteristic only of massively denatured material. Perhaps the rarely encountered 250 A-thick partition membranes of the 1-minute OsO4, 1-minute acetone dehydrated cells is the last remnant of the previous structure which is caught in the process of collapsing (Fig. 12). Furthermore, what osmium does not denature the acetone does. Polymerization at 70°C would further denature any remnants of globular protein. Fresh tissue dehydrated for 1 minute in ethylene glycol also produced a 100 Athick membrane. Thus, even in comparison to acetone and ethanol, the weak denaturing capability of ethylene glycol can cause the same denatured 100 A chloroplast membrane. This could be expected from the findings of Tanford (59), who showed that there was a limit to the concentration of ethylene glycol that y-globulin and B-lactoglobulin could be exposed to without demonstrating any unfolding of the ~-helix or producing negligible frictional changes (indicating shape). The highest concentration was 90 % ethylene glycol. Immunological reactions (indicating terti-

170

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ary structure) were affected in 7-globulin in concentrations above 60 % ethylene glycol. This explains why long exposure of dispersed cells to 100 % ethylene glycol eventually denatures the membrane protein despite previous cross-linking although the crosslinking certaintly retards the process. Ethylene glycol is therefore in no way an "inert" dehydrating agent for fresh tissue (39) when protein structure is considered. After 15 minutes of 1% glutaraldehyde cross-linking or 1-minute cross-linking in 6 % glutaraldehyde, the membrane thins to 210 A and 150 A, respectively. This parallels the findings of Quiocho and Richards (41), where the lower the concentration of glutaraldehyde, and the shorter the cross-linking time, the higher the resultant enzymatic activity of crystalline carboxypeptidase A. This implies that with longer and higher concentrations of glutaraldehyde, protein conformational changes do occur. In fact, Quiocho and Richards found that there were no changes in the X-ray diffraction pattern indicating disorder after short periods of cross-linking. Lenard and Singer (27) furthermore demonstrated that fixation of intact human blood cell membranes with 3 % glutaraldehyde resulted in a 22 % reduction in a-helix content of these membrane proteins, emphasizing the importance of a low concentration of glutaraldehyde as the cross-linking agent. It is thus concluded that as the conditions for "fixation" and dehydration of these dispersed cells favored denaturation, the chloroplast membranes changed from 270 A-thick densely packed membranes with a heterogeneous substructure (Fig. 2) to only 100 A-thick, structureless membranes often rearranged into a system of appressed disks (Fig. 11). This thin chloroplast membrane therefore represents the appearance of the membrane after denaturation of membrane proteins. This parallels the findings of Sj6strand and Barajas (56), where strong denaturing agents (heat denaturation and acidic pH) produced thin mitochondrial inner membranes. It is therefore justifiable to conclude that 1-minute cross-linking with 1% glutaraldehyde, followed by 1-minute dehydration in ethylene glycol, and 5-minute infiltration in Vestopal preserves membrane structure in a state closer to that of the native state than the other procedures that were tried. On such material, it is obvious that the substructure has the same dimensions as protein molecules known to be associated with the chloroplast membranes (Table II). The fact that morphologically 60 % of the particles are smaller than 50 A (Graph 1) corresponds to the abundance of small protein molecules associated with photosynthetic electron transport. The 40 A peak is consistent with the 40 % of the membrane protein thought to be structural protein with a molecular weight of 25 000 and a projected size of 40 A (7). The smaller bimodal peak at 80 A could morphologically represent the larger molecules known to be associated with these chloroplast membranes such as the enzymes of the fatty acid synthesis complex (7) and carboxydismutase (20). Therefore, the dimension of membrane particles corresponds to the

CHLAMY CHLOROPLAST MEMBRANES

171

dimensions of known components of the membrane or to dimensions that are reasonable for such components. Equating globular protein to this membrane particulate substructure is strengthened by evidence gained from work with the photosynthetic mutants of Cklamydomonas reinkardi. The photosynthetic mutant, ac-206, produces no active cytochrome 553 (a c-type cytochrome) (14, 15, 18, 28). All the other cytochromes are produced and function. Aside from this single gene mutation, the entire photosynthetic apparatus is intact and present. Cytochrome 553 has a molecular weight of about 13 000 and an approximate size of 30 A. The ac-206 mutation manifests itself in a morphological change in the particle histogram in the physical region where the mutant enzyme should be recorded (Graph 2). Just because the mutant makes no active cytochrome 553 does not mean that the biochemicatly functionless enzyme might not still physically insert in the membrane structure. On the other hand, the functionless enzyme could have a shape that precludes it from a membrane assembly role. At any rate, the particle histogram changes with the ac-206 mutation and suggests that this substructure may well be globular protein. The mutation results in a 5 % loss of particles smaller than 50 A and specifically in a 5 % decrease in both the 20-30 and 30-40 A intervals. Such data cannot be interpreted as the absence of cytochrome 553 molecules. This would require that 10 % of membrane proteins is cytochrome 553. A reasonable explanation would be that the absence of cytochrome 553 gives rise to secondary changes in the molecular architecture of the membrane due to modified patterns of molecular aggregation. This reorganization is then perceived as leading to an apparent decrease in the smaller size particles. Furthermore, with the sensitivity of the dispersed cell system to slight changes in the preparatory scheme, it becomes obvious that the membranes respond to parameters which are known to cause protein denaturation. For example, as the membrane thins with 10-minute cross-linking, the constituent globular subunits become larger (Graph 3). The bimodal histogram is morphologically "translated" in the direction of larger particles (20-120 A) with such increased cross-linking. This implies that the increased cross-linking had masked groups which previously were available for binding the stain, and thus the appearance of larger particles. The reduction in the thickness of the partition membrane implies a modification in the molecular architecture of the membranes presumably following conformational changes of the protein molecules. The particles were lucid areas of various sizes demarcated by a faintly staining peripheral halo which was constant in through-focus-series (Fig. 5). Such a staining pattern is compatible with what is known about globular proteins (8, 22, 32, 40). From both X-ray diffraction and thermodynamic considerations, proteins in the native state possess a dense aggregation of nonpolar amino acids in their interior and

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KRETZ~R

polar amino acids at their periphery. Certain peripheral charged amino acids would be available for binding the stain ions, while the dense hydrophobic interior would preclude penetration of the stain and would lack groups capable of binding the stain. This would result in a lucid area corresponding to the interior of globular protein molecules. The distribution of the stain is therefore compatible with the interpretation that the particulate substructure represents globular protein. Under conditions for tissue preservation that minimize protein conformational changes, no uniform layered structure could be observed in the membranes, and therefore the observations did not support the concept of a lipid bilayer as the backbone of the chloroplast membrane. Extensive areas of well-oriented lipids were unlikely due to the low birefringence of chloroplasts (32). The low polarization of fluorescence of chlorophyll (13) may indicate that these molecules share no preferred orientation with respect to some lipid bilayer. The studies of Park (34) showed that the shadowcast substructure of isolated chloroplast lamellae was constant after lipid extraction. This implied that a lipid bilayer was not a backbone structure in these chloroplast membranes. Allen (1) and Benson (3) have suggested that the specific and limited spectrum of fatty esters of surfactant lipids indicates that the lipids were associated with the membrane protein. The specificity of these fatty acid esters would not be a requirement for mere lipid-lipid interaction in a bilayer. Benson (3) proposed an intimate hydrophobic relationship between surfactant lipids and membrane protein. He stressed that lipids were not obligatorily the structural backbone of a membrane but could be essential prosthetic factors, hydrophobically related to the globular subunits. Thus, biochemically, it appeared that the lipids were not separate from the protein, but rather that the globular proteins were really globular lipoproteins. Thus the proteins determine the fate and specificity of the lipids (3, 24, 50). The model generated from these data is as follows: (a) The 270 A-thick chloroplast membrane is a three-dimensional, condensed state of globular lipropoteins. The specific and limited fatty acid esters of the membrane lipids are hydrophobically associated with specific proteins. Extensive bimolecular leaflets are nonexistent. (b) Both the surface properties of the globular proteins (represented by areas where nonpolar groups are exposed) and the shapes of the molecules are considered to favor specific types of arrangements of enzyme molecules into multienzyme complexes. Diversified enzymatic proteins are thus the membrane building blocks. In this model, the three-dimensional, specific aggregation of enzymes into multienzyme complexes is assumed to form the structural basis for the functional order and high efficiency of the chloroplast membranes. The geometrical relationships of the enzymes eliminates randomness and establishes spatial arrangements between the biochemically well-defined components. Such a condensed protein complex would

CHLAMY CHLOROPLAST MEMBRANES

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favor ordered components of such multienzyme systems as photosystem I, photosystem II, and fatty acid synthesis. (c) Proteins are held in the membrane structure mainly by hydrophobic interaction. The greater the hydrophobic interaction, the more tenaciously a protein becomes a permanent membrane component. (d) The staining at the surface of the membrane can be attributed to the polar moieties of the sulfolipids, galactolipids, and phospholipids, and to the polar amino acid side chains. (e) The chlorophyll porphyrin heads are conceptualized as being buried in the center of the 270 A-partition membrane balancing certain charged groups in this area. This proposal is based on observations that when Spurr plastic has extracted chlorophyll from the 1-minute 1% glutaraldehyde cross-linked, 1-minute ethylene glycol dehydrated thylakoid membranes, a faintly staining line develops down the center of the partition (Fig. 8). This is interpreted as being due to charged groups previously interacting with the porphyrin heads now free to bind the stain. Furthermore, with various states of denaturation (Fig. 7), this central area starts to take up the stain. This again indicates that with denaturation of protein, new charged groups are exposed which bind the stain. (f) The globular protein would dictate the orientation of the Chlorophyll phytol tails. Considering the heterogeneity of membrane proteins, this apparent random orientation of chlorophyll is a manifestation of the variations in the orientation of the chlorophyll phytol tails when intimately associated with membrane proteins. These chlorophyll-binding proteins establish numerous specific chlorophyll orientations which in toto produce the apparent "unordered" chlorophyll. Such specific chlorophyll orientations would favor energy transfer within the system. The close packing of the chlorophyll-binding protein would favor efficient energy transduction without loss of energy through fluorescence. These two factors would account for the general low fluorescence of chlorophyll in membranes as opposed to the high fluorescence of chlorophyll in solutions. (g) In order to account for strongly oriented chlorophyll at specific absorption peaks (48), it is assumed that a group of chlorophyll molecules with a pronounced orientation is associated with a specific cytochrome. In this case, the chlorophylls would be associated with the surface of this specific cytochrome. The proposed model agrees with observations of freeze-etched material where two types of bumps have been observed---a 175 A and a 110 A_ complex floating on a pebbled background (4). The relative constancy of these shapes has led others (2) to the assumption that they reflect two different complexes of enzymes. Yet the fact that only two different types of bumps are seen very likely reflects the coarseness of the technique used. At the 40 A resolution of shadowed, freeze-etched material,

174

KRETZER

only very large bumps that are large in comparison to the average dimensions of protein molecules can be observed, and small variations between different bumps on the surface of the membrane would not be discerned. Furthermore, the pebbled matrix goes unresolved. Therefore it cannot be excluded that the surface of the thylakoid membrane may well be considerably more uneven than the freeze-etched pictures show and with a great diversity of bumps. Surfaces of the proposed model would have numerous, heterogeneous bumps reflecting the underlying aggregation of protein into multi-enzyme complexes. Yet in these pictures no repeat complex units were perceived. This can be explained in three ways: (a) Let us cut a 100 A-thick section through a chloroplast membrane, which for the sake of argument, is assumed to be composed of a crystalline-packing of uniform complexes of four subunits. This section will not reveal any repeat pattern. Figure 13 depicts the most highly ordered situation that could possibly exist in a chloroplast membrane. This is not the model of the chloroplast membrane which this paper proposes. Rather tiffs diagram is the situation in which a single repeat bump (complex) has a specific orientation throughout the membrane. (This implies that the axis through the center of all these identical bumps would be uniformly oriented perpendicular to the plane of the membrane. The radial axis of all these bumps--the axis perpendicular to the former central axis--would have a constant angle of rotation.) These uniformly oriented, homogeneous bumps (equated to the 175 ~ x 100 A oblate spheres of 4 subunits often called quantasomes) would exist in a pattern most desirable for producing a repeat in the thin section. Such a pattern would be a crystalline array where such quantasomes were the only complexes in the membrane. Then the conditions would be most favorable for observing a repeat subunit in 100 A sections. Figure 13 shows that even with this most unusual situation of quantasome aggregation throughout the 270 A chloroplast thylakoids, a 100 ~ section through this membrane renders the known repeat "invisible." As the section passes through different levels of the different subunits of adjacent quantasomes the "repeat" is masked. The image is further complicated by oblique projection and superposition effects. In reality, this crystalline arrangement of 175 h-oblate spheres is the most rarely observed pattern seen in shadowcast lamellae of grana of higher plants. This diagram furthermore only includes the 175 A quantasome and says nothing about a mixture with its 110 A counterpart. In the diagram, no consideration is given to a complex aggregation of a multitude of multienzyme complexes such as the new model suggests. It is also characteristic that there is a random arrangement with respect to distances between the 175 A bumps as seen in frozen-etched material .(4, 5). Last, there seem to be different states of rotation and tilt of the 175 N bumps within the membrane

CHLAMY CHLOROPLAST MEMBRANES

175

100/~, section

270 A partition membrane

270 A partition membrane

Fro. 13. This is a hypothetical model showing that even with a crystalline array of a uniform type of complex (175 x 100 /~ with 4 subunits), a 100 A-thick section (slashed lines) masks the known repeat substructure. The complexes (quantasomes) have a constant central axis and radial axis direction. The numbers (1, 2, 3, 4) represent the 4 subunits of the quantasome. The dotted material represents the nonenzymatic protein between the uniformly layered quantasomes. This is not the model proposed by this paper. It represents the most favorable morphological situation in which to discern a repeat structure.

as reflected by fluctuations in quantasome size (160-200 A) (5). Thus, as complexity and diversity increase and approach the normal situation, the probability of seeing a repeat in thin sections of real chloroplast membranes is nil. The statistical probability of cutting a perfect tangential thin section through this diagram, if it ever existed in the cell, is infinity, considering the normal bend and tilt of chloroplast membranes. (b) Only limited areas of the obliquely cut membranes show distinct subunits. The membrane complexes would be an aggregate of many such smaller protein subunits. The infinite angles through which these complexes could be sectioned, coupled with the limited areas visible in any section, would make it impossible to perceive the numerous diversified larger aggregations. (c) Slight randomization of protein molecules during tissue preparation could also obscure these complexes. However, the previous freeze-etched pictures now accompanied by the high-

176

KRETZER

resolution, thin-section pictures presented here yield a new membrane model for the chloroplast. If however, these cross-sectioned pictures (Figs. 2-4) are blurred by being viewed through a sheet of ground glass or printed in a defocused condition, then the blurred, particulate substructure reflects larger density aggregations of protein which very well could be the quantasomes or bumps seen in freeze-etched and shadowcast material. The complexity of the model generated from these pictures is a total deviation from a homogeneous protein base structure inherent in the work of Menke (31) and Kreutz (26). Their base structure, a 40 A repeat protein, for the chloroplast membranes probably reflects the dimensions of the most common molecular components and polymers of the smaller, more numerous enzymes. Certainly a homogeneous repeat unit is far removed from the diversity inherent in Table II. In chemically fixed chloroplast material, often it has been reported by HohlHepton (19) and Weier (60, 60 that the partition membranes are the fusion product of two two-dimensional polymers of a 75 A, homogeneous protein subunit forming a partition membrane 135 ]t thick. Weier (61) found such a structure after the following fixation schemes: (a) 20 minutes 1.5 % KMnO4; (b) 3 hours 1% OsO4; (c) 30 minutes acrolein and 2 hours 1% OsO~; and (d) 60 minutes 6 % glutaraldehyde and 1-2 hours 1% OsO 4. In light of the findings of Lenard and Singer (27) that KMnO4 obliterates e-helix content and that OsO4 and glutaraldehyde, respectively, reduce 63 % and 22 % of 0~-helix content, it is doubtful if such resultant substructures have any biochemical correlation. Sjtistrand was the first to see such subdivisions in membranes of chemically fixed tissue (52) and first interpreted them as lipid micelles or globular protein (53). He later equated such homogeneous substructure in chemically fixed tissue to two alternatives: (a) partial denaturation of heterogeneous proteins to a common denominator, and/or (b) secondary aggregation of nonpolar side chains from membrane proteins which have been totally denatured and secondarily have rearranged into a uniform, globular conformation during the preparatory technique (55). Thus, chloroplast models showing a homogeneous repeat unit in a two-dimensional array are denatured artifacts of the previous in vivo structure. With such denaturation, the large 270 ]t thick, closely apposed chloroplast membranes which end abruptly as uneven packages of membrane material reorganize, form new hydrophilic surfaces from the previous hydrophobic central region of the chloroplast membrane, and separate into the classical disk stacking pattern. Such stacking is an artifact of denaturation and thus any attempt to relate "stacking profiles" to photosynthetic mutants is questionable (14, 15). The universality of the Robertson unit membranes (43, 44) can be explained by the identical effects of protein denaturation on any type of membrane.

CHLAMY CHLOROPLASTMEMBRANES

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I wish to express my thanks and gratitude to Dr F. S. Sj6strand for the privilege of studying and working in his laboratory. His invaluable advice, help, and knowledge made this study possible, Further thanks go to Miss Alice Arvin, Mrs Birgitta SjSstrand, and Miss Bibbi Wolowske for technical assistance and especially to Mr Herman Kabe for photographic help. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35.

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