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Topography and mobility of ribosomes on the surface of isolated endoplasmic reticulum of rat liver
J. Mol. Biol. (1977) 109, 589-592
LETTERS TO THE EDITOR
Topography and Mobility o f Ribosomes on the Surface of Isolated Endoplasmlc Reticulum o f Rat Liver Ribosome topography was examined on the surface of isolated lameUar rough endoplasmic reticulum. Groups of ribosomes, presumably corresponding to polysomes, were observed as circular, spiral, or parabolic structures. These were distributed fairly randomly on the membrane surface. When samples were incubated at 37~ with pancreatic ribonuclease, conditions that lead to spontaneous aggregation of free ribosomes, clustering of ribosomes to form large aggregates in the plane of the membrane was observed. Prior treatment with glutaraldehyde (5%) prevented this aggregation. It is suggested that ribosome binding components are potentially mobile, but are stabilized in native membranes forming ordered polysome conformations. Grazing sections of rough endoplasmie reticulum in intact cells demonstrate t h a t polysomes take up spiral, circular and parabolic arrangements on the membrane surface (e.g. Tara, 1967; Chua et al., 1973). Such arrangements imply t h a t either the ribosome binding sites are immobile components of the membrane or some extramembraneous factors stabilize the secondary structure of the polysome. Previous investigations of protein mobility in the endoplasmic reticulum have strongly suggested that the cytochromes P450 and bs, and their reductases are mobile in the plane of the endoplasmic retieulum membrane, (Rogers & Strittmatter, 1974a,b; Yang, 1975). We thus examined whether this was also true of the ribosome binding components. Our experiments were designed to analyze whether ribosomes could be aggregated on the surface of the endoplasmie reticulum membrane. McIntosh et al. (1975) demonstrated that free ribosomes spontaneously aggregated when incubated at 37~ for one hour, possibly as a result of partial ribonuclease digestion during the incubation. We applied this procedure to isolated rough endoplasmic reticulum and found t h a t aggregates formed on the membrane, consistent with ribosome movement on the membrane surface. In order to preserve polysome topography on the purified membranes, we isolated the reticulum in a lamellar form (Lewis & Tara, 1973) rather than a vesicular form, as considerable distortion of topography could occur during vesicle formation. Aggregate formation was assessed by determining inter-ribosome distances along membrane profiles (see legend to Fig. 3). In lamellar membranes fixed immediately after isolation, ribosomes were distributed fairly randomly along the line of the membrane, whilst polysomes exhibited spiral, circular and parabolic arrangements on the surface (Fig. 1). When incubated at 37~ in the presence of ribonuclease (bovine pancreatic enzyme, 10 ~g/ml), clear clustering of ribosomes occurred forming large aggregates separated b y nude areas of membrane (Fig. 2). The ribosome clusters, in almost all cases, followed the membrane profile, consistent with the formation of monolayer aggregates on the membrane surface. The distribution of inter-ribosome distances in the two situations is shown in t h e 589
~,
~.
FIG. 1.
FIG. 2.
FIe. 1. Surface view of rough endoplasmic reticultun fixed immediately after isolation. Arrows indicate circular, spiral, or parabolic polysome arrays. Magnification, 62,700 • The lamellar membranes were isolated from an homogenate in 0'75 M-sucrose, 2'5 m~-magnesium acetate. A pellet obtained by centrifuging the suspension at 640 g for 20 rain (50 ml tubes, MSE Mistral 6L), was resuspended in homogenizing medium to a final protein concentration of 10 mg]ml. Nuclei, mitoohondria and peroxisomes were present with the lamellar membranes. Samples were prepared for electron microscopy by settling membranes on to 0.22 /~m Millipore filters. These were fixed in osmium tetroxide (4%), glutaraldehyde (12.5%) and embedded in Spurt Resin (Spurr, 1969). Fro. 2. Aggregates of ribosomes observed on the surface of isolated membranes after incubation at 37~ for 1 h in the presence of ribonuclease (10/~g/ml). Magniflcation, 41,000 •
LETTERS
TO THE
EDITOR
591
normalized histograms in Figure 3(a) and (e). Inter-ribosome distances demonstrated a fairly broad distribution in the fixed sample. On incubation at 37~ however, the mode inter-ribosome distance decreased and deviation about the mode became smaller, consistent with aggregation of ribosomes on the surface of the endoplasmic reticulum. When incubated at 37~ in the absence of exogeneous ribonuclease, similar but less extensive aggregate formation was found (Fig. 3(d)). In both incubated cases, a considerable change in the shape of the retieulum was observed. The lamellae appeared swollen, and some fusion of membranes m a y have occurred. Such a change in morphology was also observed at 4~ but analysis of inter-ribosome distances revealed t h a t although some ribosome-free regions of membrane appeared, the ribosomes had not moved closer together to form aggregates (Fig. 3(b)). When membranes were fixed with glutaraldehyde prior to incubation at 37~ with ribonuclease, no aggregate formation took place (Fig. 3(c)). Results that were very similar to these were obtained b y quantitatively examining the distributions of ribosome density on the membrane surface, as seen in grazing sections of isolated membranes. I t is possible that aggregates were formed either by dissociation of a fraction of
2O 0
F]
. i-,-..,
40 t20
(b)j ~
0
~
r-r-F-I
~ 40 20t ( c ) ~ ~ ~ r ~ _ ~ g
0
20 0
r-"'~ m
t
r-~
120 280 440 600 7 0
9 0 40(3040000 (,~)
Inter-ribosome distance
Fro. 3. Normalized histograms demonstrating the distribution of inter-ribosome distances. These were estimated after plotting ribosome positions on cellulose acetate paper a n d enlarging to a final magnification of 300,000 to 400,000 times. A t least 200 estimations were made for each treatment. (a) Membranes fixed with glutaraldehyde (5%) a t 4~ a n d left a t 4~ for 1 h prior to processing for electron microscopy. (b) Membranes left a t 4~ for 1 h a n d t h e n fixed w i t h glutaraldehyde (5%). (c) Membranes fixed w i t h glutaraldehyde (5%) a t 4~ a n d t h e n i n c u b a t e d for 1 h a t 37~ (d) Membranes i n c u b a t e d a t 37~ for 1 h w i t h o u t prefixation. (e) Membranes incubated a t 37~ with ribonuclease (10 ~g/m]) for 1 h prior to processing.
592
G. P A R R Y , C. D A V I E AND D. J. W I L L I A M S
ribosomes from the membrane and subsequent rebinding to bound ribosomes, or by m o v e m e n t of the ribosome binding site in the plane of the membrane. I t was found t h a t incubation of lame]lar membranes at 25~ resulted in loss of some ribosomes (Parry & Williams, unpublished observations). However, it is likely t h a t if significant rebinding occurred it would result in the formation of large clusters where all the ribosomes were not directly associated with the m e m b r a n e but were associated directly with bound ribosomes (l~IcIntosh et al., 1975). I t was clear t h a t a large proportion of the aggregates followed the line of the membrane, implying t h a t aggregate formation followed m o v e m e n t of the ribosome binding site in the m e m b r a n e lipid, l~Ioreover, the mode inter-ribosome distance in the aggregated state was approximately 240 ~ ; ribosome diameter measured under similar conditions to those used here is 250 A (Spirin, 1969), consistent with the ribosomes forming two-dimensional arrays on the surface with all ribosomes in contact with adjacent ribosomes. I t thus appears t h a t ribosome binding sites are mobile in the m e m b r a n e lipid but are stabilized in the native m e m b r a n e such t h a t spiral, circular and parabolic polysome conformations can be formed on the m e m b r a n e surface. The experiments do not give a n y information concerning the nature of the stabilizing factors. I t is possible t h a t ribonuclease action results in breakage of a stabilizing m R N A molecule, but as such action is also probably required to create "sticky" ribosomes t h a t have potential to aggregate, no definitive conclusions can be reached. I t appears t h a t these conclusions are consistent with those of Ojakian & Sabatini (1975), who have analyzed ribosome distribution in microsomal membranes using freeze-etch techniques. The mobility of ribosome binding components is thus suggested b y both transmission electron microscopy of lamellar endoplasmic m e m b r a n e and freeze-etch microscopy of microsomal membranes. We thank Dr P. R. McIntosh and Prof. B. R. Rabin for helpful discussion of this work, and for informing us of their unpublished results. We thank the Medical Research Council and the Nuffleld Foundation for financial support. Dept. of Biochemistry University College London Gower Street, London W.C.1, England.
GORDON PARRY t CABOT.DAV~ DAVID J. WILLIA~S
Received 1 June 1976, and in revised form 27 October 1976 REFERENCES Chua, N., Blobel, G., Siekevitz, P. & Palade, G. E. (1973). Prec. Nat. Acad. Sci., U.S.A. 7{), 1554-1558. Lewis, J. & Tata, J. R. (1973). J. Cell Sci. 13, 447-460. McIntosh, P. R., Clark, R. F. & Rabin, B. R. (1975). _~EBS Letters, 6{}, 190-196. Ojakian, G. & Sabatini, D. (1975). J. Cell. Biol. {}7, 314a. Rogers, M. J. & Strittmatter, P. (1974a). J. Biol. Chem. 249, 895-900. Rogers, M. J. & Strittmatter, P. (1974b). J. Biol. Chem. 249, 5565-5569. Spirin, A. S. (1969). Prog. Biophys..~lol. Biol. 19, 135-174. Spurr, A. R. (1969). J. Ultrastruct. Res. 2{}, 31-43. Tata, J. R. (1967). Biochem. J. 1{}5, 783-801. Yang, C. S. (1975). F E B S Letters, 54, 61-64. t Present address: Laboratory of Chemical Biodynamics, Lawrence Berkeley Laboratory, University of California, Berkeley, Calif. 94720, U.S.A.
LETTERS TO THE EDITOR
Topography and Mobility o f Ribosomes on the Surface of Isolated Endoplasmlc Reticulum o f Rat Liver Ribosome topography was examined on the surface of isolated lameUar rough endoplasmic reticulum. Groups of ribosomes, presumably corresponding to polysomes, were observed as circular, spiral, or parabolic structures. These were distributed fairly randomly on the membrane surface. When samples were incubated at 37~ with pancreatic ribonuclease, conditions that lead to spontaneous aggregation of free ribosomes, clustering of ribosomes to form large aggregates in the plane of the membrane was observed. Prior treatment with glutaraldehyde (5%) prevented this aggregation. It is suggested that ribosome binding components are potentially mobile, but are stabilized in native membranes forming ordered polysome conformations. Grazing sections of rough endoplasmie reticulum in intact cells demonstrate t h a t polysomes take up spiral, circular and parabolic arrangements on the membrane surface (e.g. Tara, 1967; Chua et al., 1973). Such arrangements imply t h a t either the ribosome binding sites are immobile components of the membrane or some extramembraneous factors stabilize the secondary structure of the polysome. Previous investigations of protein mobility in the endoplasmic reticulum have strongly suggested that the cytochromes P450 and bs, and their reductases are mobile in the plane of the endoplasmic retieulum membrane, (Rogers & Strittmatter, 1974a,b; Yang, 1975). We thus examined whether this was also true of the ribosome binding components. Our experiments were designed to analyze whether ribosomes could be aggregated on the surface of the endoplasmie reticulum membrane. McIntosh et al. (1975) demonstrated that free ribosomes spontaneously aggregated when incubated at 37~ for one hour, possibly as a result of partial ribonuclease digestion during the incubation. We applied this procedure to isolated rough endoplasmic reticulum and found t h a t aggregates formed on the membrane, consistent with ribosome movement on the membrane surface. In order to preserve polysome topography on the purified membranes, we isolated the reticulum in a lamellar form (Lewis & Tara, 1973) rather than a vesicular form, as considerable distortion of topography could occur during vesicle formation. Aggregate formation was assessed by determining inter-ribosome distances along membrane profiles (see legend to Fig. 3). In lamellar membranes fixed immediately after isolation, ribosomes were distributed fairly randomly along the line of the membrane, whilst polysomes exhibited spiral, circular and parabolic arrangements on the surface (Fig. 1). When incubated at 37~ in the presence of ribonuclease (bovine pancreatic enzyme, 10 ~g/ml), clear clustering of ribosomes occurred forming large aggregates separated b y nude areas of membrane (Fig. 2). The ribosome clusters, in almost all cases, followed the membrane profile, consistent with the formation of monolayer aggregates on the membrane surface. The distribution of inter-ribosome distances in the two situations is shown in t h e 589
~,
~.
FIG. 1.
FIG. 2.
FIe. 1. Surface view of rough endoplasmic reticultun fixed immediately after isolation. Arrows indicate circular, spiral, or parabolic polysome arrays. Magnification, 62,700 • The lamellar membranes were isolated from an homogenate in 0'75 M-sucrose, 2'5 m~-magnesium acetate. A pellet obtained by centrifuging the suspension at 640 g for 20 rain (50 ml tubes, MSE Mistral 6L), was resuspended in homogenizing medium to a final protein concentration of 10 mg]ml. Nuclei, mitoohondria and peroxisomes were present with the lamellar membranes. Samples were prepared for electron microscopy by settling membranes on to 0.22 /~m Millipore filters. These were fixed in osmium tetroxide (4%), glutaraldehyde (12.5%) and embedded in Spurt Resin (Spurr, 1969). Fro. 2. Aggregates of ribosomes observed on the surface of isolated membranes after incubation at 37~ for 1 h in the presence of ribonuclease (10/~g/ml). Magniflcation, 41,000 •
LETTERS
TO THE
EDITOR
591
normalized histograms in Figure 3(a) and (e). Inter-ribosome distances demonstrated a fairly broad distribution in the fixed sample. On incubation at 37~ however, the mode inter-ribosome distance decreased and deviation about the mode became smaller, consistent with aggregation of ribosomes on the surface of the endoplasmic reticulum. When incubated at 37~ in the absence of exogeneous ribonuclease, similar but less extensive aggregate formation was found (Fig. 3(d)). In both incubated cases, a considerable change in the shape of the retieulum was observed. The lamellae appeared swollen, and some fusion of membranes m a y have occurred. Such a change in morphology was also observed at 4~ but analysis of inter-ribosome distances revealed t h a t although some ribosome-free regions of membrane appeared, the ribosomes had not moved closer together to form aggregates (Fig. 3(b)). When membranes were fixed with glutaraldehyde prior to incubation at 37~ with ribonuclease, no aggregate formation took place (Fig. 3(c)). Results that were very similar to these were obtained b y quantitatively examining the distributions of ribosome density on the membrane surface, as seen in grazing sections of isolated membranes. I t is possible that aggregates were formed either by dissociation of a fraction of
2O 0
F]
. i-,-..,
40 t20
(b)j ~
0
~
r-r-F-I
~ 40 20t ( c ) ~ ~ ~ r ~ _ ~ g
0
20 0
r-"'~ m
t
r-~
120 280 440 600 7 0
9 0 40(3040000 (,~)
Inter-ribosome distance
Fro. 3. Normalized histograms demonstrating the distribution of inter-ribosome distances. These were estimated after plotting ribosome positions on cellulose acetate paper a n d enlarging to a final magnification of 300,000 to 400,000 times. A t least 200 estimations were made for each treatment. (a) Membranes fixed with glutaraldehyde (5%) a t 4~ a n d left a t 4~ for 1 h prior to processing for electron microscopy. (b) Membranes left a t 4~ for 1 h a n d t h e n fixed w i t h glutaraldehyde (5%). (c) Membranes fixed w i t h glutaraldehyde (5%) a t 4~ a n d t h e n i n c u b a t e d for 1 h a t 37~ (d) Membranes i n c u b a t e d a t 37~ for 1 h w i t h o u t prefixation. (e) Membranes incubated a t 37~ with ribonuclease (10 ~g/m]) for 1 h prior to processing.
592
G. P A R R Y , C. D A V I E AND D. J. W I L L I A M S
ribosomes from the membrane and subsequent rebinding to bound ribosomes, or by m o v e m e n t of the ribosome binding site in the plane of the membrane. I t was found t h a t incubation of lame]lar membranes at 25~ resulted in loss of some ribosomes (Parry & Williams, unpublished observations). However, it is likely t h a t if significant rebinding occurred it would result in the formation of large clusters where all the ribosomes were not directly associated with the m e m b r a n e but were associated directly with bound ribosomes (l~IcIntosh et al., 1975). I t was clear t h a t a large proportion of the aggregates followed the line of the membrane, implying t h a t aggregate formation followed m o v e m e n t of the ribosome binding site in the m e m b r a n e lipid, l~Ioreover, the mode inter-ribosome distance in the aggregated state was approximately 240 ~ ; ribosome diameter measured under similar conditions to those used here is 250 A (Spirin, 1969), consistent with the ribosomes forming two-dimensional arrays on the surface with all ribosomes in contact with adjacent ribosomes. I t thus appears t h a t ribosome binding sites are mobile in the m e m b r a n e lipid but are stabilized in the native m e m b r a n e such t h a t spiral, circular and parabolic polysome conformations can be formed on the m e m b r a n e surface. The experiments do not give a n y information concerning the nature of the stabilizing factors. I t is possible t h a t ribonuclease action results in breakage of a stabilizing m R N A molecule, but as such action is also probably required to create "sticky" ribosomes t h a t have potential to aggregate, no definitive conclusions can be reached. I t appears t h a t these conclusions are consistent with those of Ojakian & Sabatini (1975), who have analyzed ribosome distribution in microsomal membranes using freeze-etch techniques. The mobility of ribosome binding components is thus suggested b y both transmission electron microscopy of lamellar endoplasmic m e m b r a n e and freeze-etch microscopy of microsomal membranes. We thank Dr P. R. McIntosh and Prof. B. R. Rabin for helpful discussion of this work, and for informing us of their unpublished results. We thank the Medical Research Council and the Nuffleld Foundation for financial support. Dept. of Biochemistry University College London Gower Street, London W.C.1, England.
GORDON PARRY t CABOT.DAV~ DAVID J. WILLIA~S
Received 1 June 1976, and in revised form 27 October 1976 REFERENCES Chua, N., Blobel, G., Siekevitz, P. & Palade, G. E. (1973). Prec. Nat. Acad. Sci., U.S.A. 7{), 1554-1558. Lewis, J. & Tata, J. R. (1973). J. Cell Sci. 13, 447-460. McIntosh, P. R., Clark, R. F. & Rabin, B. R. (1975). _~EBS Letters, 6{}, 190-196. Ojakian, G. & Sabatini, D. (1975). J. Cell. Biol. {}7, 314a. Rogers, M. J. & Strittmatter, P. (1974a). J. Biol. Chem. 249, 895-900. Rogers, M. J. & Strittmatter, P. (1974b). J. Biol. Chem. 249, 5565-5569. Spirin, A. S. (1969). Prog. Biophys..~lol. Biol. 19, 135-174. Spurr, A. R. (1969). J. Ultrastruct. Res. 2{}, 31-43. Tata, J. R. (1967). Biochem. J. 1{}5, 783-801. Yang, C. S. (1975). F E B S Letters, 54, 61-64. t Present address: Laboratory of Chemical Biodynamics, Lawrence Berkeley Laboratory, University of California, Berkeley, Calif. 94720, U.S.A.