A 20S Particle Ubiquitous from Yeast to Human

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A 20S Particle Ubiquitous from Yeast to Human

ARRIGO, André-Patrick, et al.

Abstract

We have purified and characterized a particle sedimenting at 20S from the postribosomal fraction of yeast, wheat germ,Drosophila melanogaster tissue culture cells, chicken embryo fibroblasts, rabbit reticulocyte lysate, and HeLa cells. Most of the protein constituents of the 20S particle have molecular weights of 20–35 kd and differ between species; however, some do have similar molecular weights and isoelectric points, suggesting they are related. Several low-molecular-weight RNAs, distinct from tRNAs, co-purify with the particle isolated from all these species and show increasingly more complex patterns ascending the arbitrary order from yeast to human (yeast, plant, insect, bird, and mammals). InDrosophila, we present evidence that these small RNAs are tightly associated with this 20S structure.

ARRIGO, André-Patrick, et al . A 20S Particle Ubiquitous from Yeast to Human. Journal of Molecular Evolution , 1987, vol. 25, no. 2, p. 141-150

DOI : 10.1007/BF02101756

Available at:

http://archive-ouverte.unige.ch/unige:149376

Disclaimer: layout of this document may differ from the published version.

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J Mol Evol (1987) 25:141-150

Journal of

Molecular Evolution

@ Springer-Verlag New York Inc. 1987

A 20S Particle Ubiquitous from Yeast to Human

A.-P. Arrigo, 1,2 M. Simon, 2 J.-L. Darlix, 3 and P.-F. Spahr 2

' Cold Spring Harbor Laboratory, P.O. Box 100, Cold Spring Harbor, New York 11724, USA

: Department of Molecular Biology, University of Geneva 30, Quai Ernest Ansermet, 1211 Geneva-4, Switzerland 3 CRBGC-CNRS, Universit6 Paul Sabatier, 31062 Toulouse, France

Summary. We have purified and characterized a particle sedimenting at 20S from the postribosomal fraction of yeast, wheat germ, Drosophila melano- gaster tissue culture cells, chicken embryo fibroblasts, rabbit reticulocyte lysate, and HeLa cells.

Most of the protein constituents of the 20S particle have molecular weights of 20-35 kd and differ between species; however, some do have similar molecular weights and isoelectric points, suggesting they are related. Several low-molecular-weight RNAs, distinct from tRNAs, co-purify with the particle isolated from all these species and show increasingly more complex patterns ascending the arbitrary order from yeast to human (yeast, plant, insect, bird, and mammals). In Drosophila, we present evidence that these small RNAs are tightly associated with this 20S structure.

Key words: 20S particle -- Prosome -- Evolution -- Small cytoplasmic RNAs -- Heat shock proteins

Introduction

Particles of unknown function, sedimenting at about 20S on a sucrose gradient, have been described in several eukaryotic cells. Such complexes have been found in the cytoplasm of human cells and pea seedlings (Shelton et al. 1970), mammalian ceils (Harris 1970; Narayan and Round 1973; Smulson 1974;

Harmon et al. 1983; Schmidt et al. 1984; Martin de Offprint requests to: A.-P. Arrigo at Cold Spring Harbor Labo- ratory

Abbreviations: SDS, sodium dodecyl sulfate; PMSF, phenyl- methanesulfonyl ttuoride

Saet al. 1986), and duck erythroblasts (Schmidt et al. 1984; Martin de Saet al. 1986). Recently, we (Arrigo et al. 1985) and others (Schuld and Kloetzel 1985) have described these particles in Drosophila melanogaster KC tissue culture cells. In Xenopus oocytes similar particles were also found in the nu- cleus (Hugle et al. 1983; Kleinschmidt et al. 1983).

In all cases, their shape, as determined by electron microscopy, appears as a "hollow cylinder" made of four stacked annuli. Biochemical studies (Har- mon et al. 1983; Kleinschmidt et al. 1983; Schmidt et al. 1984; Arrigo et al. 1985; Schuld and Kloetzel 1985) have shown that these particles are made of several proteins with molecular weights in the range of 20-35 kd and some of higher molecular weight.

However, differences have been reported with re- spect to the dimensions of this type of structure and the number of its protein constituents. Small RNAs have been found to co-purify with the particles isolated from human cells (Narayan and Round 1973), mouse and duck erythroblasts (Schmidt et al. 1984), and D. melanogaster tissue culture ceils (Arrigo et al. 1985; Schuld and Kloetzel 1985). The name

"prosome" has been proposed for this type of particle (Schmidt et al. 1984) and is used in this study.

Here we present a comparative analysis of the cytoplasmic 20S prosomal particles isolated from yeast, wheat (wheat germ), D. rnelanogaster (tissue culture cells), chicken (embryo fibroblasts), rabbit (reticulocyte lysate), and human (HeLa cells). In this paper, we show that from yeast to human, the shape of the prosome has been conserved and that its dimensions, although not identical in all species, are very similar. The protein constituents of the prosome are not identical between species, but form a

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family ofpolypeptides with molecular weights ranging between 20 and 35 kd. Some of the polypeptides observed in the different species have similar molecular weights and isoelectric points, suggesting that they are highly related. In each species analyzed, small RNAs co-purify with the prosome and appear relatively well conserved throughout evolution. In one species, D. melanogaster, evidence is presented suggesting that these small RNAs are tightly associated with the prosomal structure. These obser- vations are discussed in reference to the possible role played by this enigmatic structure.

Materials and Methods

Cell Cultures. 1) Yeast: Saccharomyces cerevisiae, strain YNN27 (Stinchcomb et al. 1980), was grown at 30~ with vigorous agi- tation in YPD medium. 2) Wheat: Wheat germ (a gift from J.W.

Davies) was used as starting material. 3) Fly: D. melanogaster tissue culture cells KCI 61 were grown as described previously (Arrigo et al. 1985). 4) Chicken embryo fibroblasts were grown as described previously (Arrigo et al. 1983). 5) Rabbit: Crude rabbit reticulocyte lysate, not treated with nuclease (Clinical Con- venience Product, Inc., Madison, WI), was used as starting material. 6) Human: HeLa cells were grown at 37"C in 60-mm plastic dishes (Falcon) in Dulbecco modified Eagle medium supple- mented with 10% fetal calf serum in temperature-controlled hu- midified incubators (5% CO2).

Cell Lysis. 1) Yeast: Lysis was performed at 0~ in 10 mM Tris-HCl (pH 7.5), l0 mM NaCl, 5 mM MgCl2, 1 mM/~-mercaptoethanol, and 1 mM PMSF in the presence of a similar volume of glass beads (0.5 mm in diameter). The preparation was vortexed twice for 2.5 rain. NP-40 (0.5% final concentration) was added, and the mixture was vortexed again and centrifuged for 10 min at 16,000 x g. The supernatant was further centrifuged for 16 h at 150,000 x g in an A321 rotor (International Centri- fuge). The resulting pellet was used in the isolation of the 20S particle. 2) Wheat germ was ground with the same weight of glass beads (0.2 mm in diameter) in a buffer (five times the weight of the wheat germ) containing 20 mM Tris-HC1 (pH 7.5), 100 mM KC1, 1 mM MgCI2, 2 mM CaC12, 6 mM/3-mercaptoethanol, and 1 mM PMSF. Cells were ground for approximately 1 min at 0*C.

The mixture was then centrifuged exactly as described above for the yeast, and again the pellet used for the isolation of the 20S particle. 3) Drosophila tissue culture cells were lysed as described previously (Arrigo et al. 1985) and treated as described above.

4) Chick embryo fibroblasts were lysed by repeated homogenization in 10 mM Tris-HCl (pH 7.5), I0 mM NaC1, 10 mM MgC12, 0.5~ NP-40, 1 mM B-mercaptoethanol, and 1 mM PMSF and the lysis mixture was clarified for 10 min at 16,000 x g. The 20S particles were isolated either directly from this supernatant, or after it had been concentrated by further centrifugation at 150,000 x g for 16 h. 5) Rabbit reticulocyte lysate was spun for 16 h at 150,000 x g as above, and the pellet was used as starting material. 6) HeLa cells were lysed by repeated homogenization as described above for chicken embryo fibroblasts. The lysis mixture was clarified for 10 rain at 16,000 x g and the supernatant treated as above.

Purification of the 20S Particle. Pellets from the 150,000 x g centrifugation were resuspended in buffer A [10 mM Tris-HC1 (pH 7.5), 10 mM NaC1, 10 mM MgCI2, 1 mM B-mercaptoethanol]

and centrifuged on 0.5-1 M sucrose gradients (Schwarzmann;

RNase-free) in 10 mM Tris-HC1 (pH 7.5), 10 mM NaC1, and 5 mM MgCI2, for 17 h at 35,000 rpm in an SB283 rotor (Inter- national Centrifuge). The 16,000 x g supernatant was sedimented the same way when the concentration step was omitted. The fractions containing the 20S particle were pooled, diluted 1:1 with buffer A, and rerun as above. The purification by sucrose gradient was repeated a third time in presence of 0.5 M NaC1.

Polyacrylamide Gel Analysis of Proteins. Proteins were precipitated with 20% cold TCA (final concentration) for 3 h at 0~

and collected by centrifugation at 16,000 x g for 30 min. The protein pellet was washed with acetone, dried, resuspended in sample buffer, and heated to 100*C for 1 min. Samples were analyzed by SDS-polyacrylamide gel electrophoresis (12.5% acrylamide, 0.9% bis-acrylamide) as described by Laemmli (1970).

Gels were fixed, stained with Coomassie blue, destained, and photographed.

Two-Dimensional Gel Electrophoresis of Proteins. Two-dimensional gel electrophoresis was performed according to O'Farrell (1975) as modified by Khandjian and Trifler (1983).

The 20S particles pelleted for 16 h at 150,000 x g were resuspended in 40 tzl of 10 mM Tris-HC1 (pH 7.4) containing 100 ~tg of pancreatic RNase and incubated for 30 rain at room temperature. Samples were then mixed with the urea-containing lysis buffer and loaded at the anodic end of the first-dimension gel containing 2% (w/v) ampholytes (pH 5-7) (LKB-Produkter AB, Bromma, Sweden). Electrofocusing was for 5000 V x h. Second- dimension SDS gel electrophoresis and protein staining were as described above.

Nondenaturing Gel Electrophoresis. Composite gels of 2% acrylamide--0.5% agarose in Tris-borate buffer (pH 8.3) (Dahlberg et al. 1969) were used as previously described (Arrigo et al. 1985).

Analysis of RNA. The 20S particles were digested for 1 h at 37~ with 1 mg/ml proteinase K in the presence of 0.1% SDS.

RNA was extracted with phenol-chloroform, precipitated by eth- anol, and labeled at the 3' end with [32p]pCp (R.C. Amersham, 3000 Ci/mmol) and RNA ligase (Pharmacia) as described by England et al. (1980) and modified by Keith (1983). The labeled RNAs were then resolved in 12% polyacrylamide gels containing 7 M urea (Darlix et al. 1979). Autoradiography was done at -70~ using Fuji x-ray film with the aid of Ilford fast tungstate intensifying screens. T 1 fingerprints were performed as described (Arrigo et al. 1983). RNAs were denatured and digested with TI RNase (Calbiochem). T1 oligonucleotides were labeled with [3,:zP]ATP (R.C. Amersham, 3000 Ci/mmol) and T4 polynu- cleotide kinase (Biotec) and were resolved by two-dimensional gel electrophoresis as described by de Wachter and Fiers (1972), except that the second dimension was run in 50 mM Tris-borate (pH 8.3). Autoradiography was performed as above but at 4"C.

5':2P-labeled T1 oligonucleotides were cut out, eluted, and purified further on a 20% acrylamide sequencing polyacrylamide gel in 7 M urea (Maxam and Gilbert 1977). The purified 5'-32p - labeled T 1 oligonucleotides were sequenced following the method described by Donis-Keller et al. (1977) and Simonscists et al.

(1977).

Electron Microscopy. A drop of the purified fraction containing the 20S particle was adsorbed onto freshly glow-discharged carbon-coated grids. After absorbing the excess of the purified fraction with filter paper, the grids were rinsed with water and stained with 0.1% uranyl acetate before being air dried. Electron micrographs were taken with a Philips 300 electron microscope at 80 kV. The size of the particles was measured on 400,000 x enlarged prints. Mean dimensions and standard deviations were determined.

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Fig. 1. Structural characterization of the 20S prosomal particles by electron microscopy. The 20S panicles purified from yeast, wheat, Drosophila, chicken, rabbit, and human were analyzed by electron microscopy as described in Materials and Methods. Low- magnification views showing the distribution of the three types of structures in the different species analyzed (rounded with or without a central black zone and cylindrical) are presented. Bar = 50 nm

Results

Electron Microscopy Analysis

The 20S cytoplasmic prosomal structure was isolated as described in Materials and Methods from yeast, wheat, fly, chicken, rabbit, and human ceils.

Analyzing these samples by electron microscopy (Fig.

1) reveals a high degree of structural conservation in the prosome from yeast to human. No 20S prosomal structure has been found in Escherichia coli extracts (data not shown); we would like to suggest that the prosome is specific to eukaryotes. In each

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Fig. 2. Intermediate magnification of arbitrarily selected electron micrographs showing the different morphologies of the 20S prosomal particles in the different species analyzed. Bar = 20 nm. Shape and size of the particles are well conserved. See Table 1 for a statistical analysis of the dimensions of these structures. The cylindrical structures present four suhunits that could he stacks of the rounded particles

organism, three types of structures were observed (Figs. 2 and 3): 1) The first is hexagonal with a dense central zone. 2) The second type is cylindrical and appears to be a composite stack of four substructures of a diameter similar to the hexagonal particle. 3) The third type is also hexagonal but without a central heavily stained zone. This structure is probably identical to the first one, but possibly having ad- ditional polypeptides filling the central zone. High magnifications (Fig. 3) indicate that 1) the hexagonal structure is probably made of six subunits arranged hexagonally around a central zone that does not stain with uranyl acetate and 2) each of the four substructures of the cylinder is probably made of several subunits that may be single polypeptides.

The two types of hexagonal structures (with or without a central black zone) are either isolated substructures of the cylinder or more probably (see below, Fig. 4B) standing cylinders viewed from either above or below. Table 1 shows the percentage and the size of the three types of structure in the different species analyzed. In every species the hexagonal structures are the most abundant. Between species, the mean diameter of the structures varies between 13 and 14 nm, while the mean length is between 17 and 19 nm. Moreover, according to statistical analysis (Table 1), the size variation of the prosome among the six different species is similar to that observed within samples from the same species.

These results indicate a high degree of conservation of the size of the prosome.

Fig. 3. Higher magnification (enlargements 800,000 x; bar = 10 nm) of arbitrarily selected electron micrographs of the 20S prosomal particles. A The rounded particles reveal a hexagonal morphology composed of six subunits arranged around a dense central zone; B the cylindrical particles are made of four sub-

c--

structures, which are themselves made of several subunits; C the rounded particles without a dense central zone also reveal a hexagonal morphology

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Fig. 4. Gel electrophoresis of the protein constituents of the 20S prosomai particles purified by three runs in sucrose gradients. A SDS polyacrylamide gel; B nondenaturing gel run at pH 8.3 (Dahlberg et al. 1969). The Drosophila 20S prosome has an apparent molecular weight of about 7.5 • 10 ~ in nondenaturing gels (see Arrigo et al. 1985). Coomassie blue-stained gels are presented, a Yeast;

b wheat; c Drosophila; d chicken; e rabbit; f human

Protein Constituents Analysis of the 20S Prosomal Particle

In contrast to its u b i q u i t o u s shape, the protein composition of the p r o s o m e is not identical when the various species are analyzed (Fig. 4A). In each case, however, the proteins of the p r o s o m e migrate in SDS gels as a family of polypeptides with molecular weights ranging between 20 a n d 35 kd. In some species high-molecular-weight proteins are present;

they are not believed to be constituents of the prosome because (as, for example, in Drosophila) m o s t of t h e m can be r e m o v e d by h y d r o p h o b i c c h r o m a - tography w i t h o u t drastically affecting the shape of

the prosome (see below, Figs. 8 a n d 9). In yeast, prosomal preparations contain one m a j o r polypeptide of 26 kd although weaker bands (20-35 kd) are also seen (see the t w o - d i m e n s i o n a l gel analysis in Fig. 5 for a better resolution of these proteins). T h e 20S prosomal particle of each species was further analyzed in n o n d e n a t u r i n g gels run at pH 8.3 (see Materials and Methods). Figure 4B shows all the prosomes migrating as a single sharp band, suggesting that only one type of structure is present in every species. This would support the proposal that the hexagonal and cylindrical particles described above represent different views of the same structure. Since the overall shape of the prosome r e m a i n s Table 1. Characteristics of the 20S prosomal particles

Yeast Wheat Drosophila Chicken Rabbit Human

Hexagonal particle with central dark zone

Percentage 53 47 59 49 45 49

Diameter (nm) 13 • 1.5 14.5 + 1.5 14 _+ 1 13.5 _+ 1 13 • I 13 + 1

Diameter of dark zone (nm) 3.5 • 1 3.5 _+ 1 3.5 + 1 3.5 • 1 3.5 • 1 3.5 • 1 Hexagonal

Percentage 33 30 31 34 50 45

Diameter (nm) 13 • 2.5 14 + 2 14 _ 1 14 _ 3 13 • 1 13 + 1

Cylindrical

Percentage 14 23 10 17 5 6

Diameter (nm) 13 • 1 14 _+ 1 14 _ 1 14 • 1 13 • 1 13 • 1

Length (nm) 18 • 1 19 _ 1 18.5 • 1 18 • 1 17 _+ 1 17 +_ 1

The percentages of the three types of 20S structure in the different species analyzed were determined from 200,000 x enlarged prints.

Average percentages from several independent determinations are presented. The dimensions of the 20S structures were measured on 400,000 x enlarged prints and are indicated in nanometers. The mean values and the standard errors (for each species n = 12, i.e., six determinations in two separate experiments) were rounded off to the nearest half unit (nm) (i.e., 14.2 becomes 14.0 and 14.3 becomes 14.5)

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Fig. 5. Two-dimensional gel analysis of the 20-35-kd proteins of the prosome. The prosomes isolated from the different species were analyzed in two-dimensional gels as described in Materials and Methods. Coomassie blue staining of the SDS gels is presented, a Yeast; b wheat; c Drosophila; d chicken; e human. 0, 26-kd protein of yeast homologous to the yeast heat-shock protein HSP26 (S. Lindquist, personal communication); v, proteins analogous between yeast and wheat; v, proteins analogous between wheat and Drosophila; .1., proteins analogous between Drosophila and chicken; v, proteins analogous between chicken and human

unchanged from yeast to human, the gradient of decreasing electrophoretic migration observed un- der nondenaturing conditions possibly reflects a gradual reduction in the negative charge of the prosome in the arbitrary pathway yeast-plant-insect- bird and mammals. This view is supported by the finding that the prosomes isolated from the two m a m m a l i a n species (human and rabbit) have the same migration.

Analyzing the protein constituents of the prosome further by two-dimensional gel electrophoresis reveals (Fig. 5) a complex pattern of proteins with isoelectric points between pH 4.6 and 7.6, which is different between species. In yeast, one polypeptide of 26 kd (black arrow, Fig. 5a) is far more abundant than the 19 other 20-35-kd proteins. In wheat,

Fig. 6. Analysis of the RNA associated with the prosome purified from different species. The prosome was isolated by three successive runs of sucrose gradients as described in Materials and Methods. RNA sedimenting at 20S together with the prosome was purified, labeled in vitro at its 3' end with [32p]pCo, and analyzed in 7 M urea-8% polyacrylamide gels as described in Materials and Methods. An autoradiograph of the gel is presented, a and g E. coli tRNA as size marker; b yeast; e wheat;

d Drosophila; e chicken; f human

12 major polypeptides are seen; there are 18 in Dro- sophila, 18 in chicken, and 12 in HeLa cells. The protein pattern of rabbit prosome is similar to that observed in humans (not shown). Some of the polypeptides observed have identical molecular weights and isoelectric points, which suggests that they are related. From such an analysis, one can detect homologous polypeptides of the prosome in the different species from yeast to h u m a n (see the arrows in Fig. 5a-e): in each case some of the polypeptides of the prosome of the earlier species remain while new ones appear. The classification was performed on the "arbitrary pathway" of plant-insect-bird and mammals. These results suggest that, between species, some prosomal proteins are related, probably in order to maintain a conserved shape of the particle during evolution.

RNA Constituent Analysis

It has been reported that in duck and mouse erythroblasts (Schmidt et al. 1984) and in D. melanogaster (Arrigo et al. 1985; Schuld and Kloetzel 1985) RNAs of small size co-sediment with the prosome. Figure 6 shows, for the different species described above, the co-sedimentation with the prosome (after three runs in sucrose gradients) of R N A labeled at the 3'

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Fig. 7. Comparison of the complexity of the RNA associated with the prosome isolated from different species. The RNA co-purifying with the prosomes was analyzed by T1 fingerprints as described in Materials and Methods. a Yeast tRNA as control; b-f Prosomal

RNA: b yeast; c wheat; d Drosophila; e chicken; f human. V, TI oligonucleotides conserved between yeast and the other species;

V,T1 oligonucleotides conserved between Drosophila and chicken; Iv, TI oligonucleotides conserved between chicken and human end with

[32

P]pCp. In each case, several small RNAs

that migrate slightly differently from tRNAs are observed. Similar to what has been observed in duck erythroblasts by Schmidt et al. (1984), only two major small RNAs co-purify with the prosome isolated from chicken fibroblasts (Fig. 6e). In wheat, 5S RNA is also found (Fig. 6c) and may represent a contam- ination. Our previous work (Arrigo et al. 1985) using two-dimensional gel analysis and sequencing studies has revealed at least five different RNAs associated to the Drosophila prosome. As an approach to com- pare the complexity of the RNAs of the prosome between species, T1 fingerprint analysis was performed. Figure 7 shows that in each case the small RNAs of the 20S particle are resolved into several well-defined T 1 oligonucleotides that are clearly different from those of yeast tRNAs. The sequence complexity of the RNA associated with the 20S particle gradually increases from yeast to human. How- ever, several common T 1 oligonucleotides are also observed between different species; for instance, the four major yeast oligonucleotides (white arrows) are recovered in wheat and Drosophila while they are gradually lost in chick and rabbit, and only one

remains in humans (similar T 1 fingerprints are observed in rabbit and human; data not shown). Sev- eral chicken T 1 oligonucleotides (black arrows) are recovered in humans, but are not found in the other species.

Sequence analysis confirms and extends this evo- lutionary trend. The sequences of the yeast and Dro- sophila oligonucleotides I (see Fig. 7b and d) are identical (5'-G-U-A-A-C-A-A) and so are the sequences of oligonucleotides III (5'-G-C-C-A-C).

Only one change of base position was found between the yeast and Drosophila oligonucleotides II (5'-G- U-C-A-C-A-A and 5'-G-U-C-C-A-A-A, respective- ly).

These results indicate that most of the small RNAs co-purifying in sucrose gradients with the prosome are distinct from the bulk oftRNAs and are related between species.

Chromatography of the Drosophila 20S Prosomal Particle

To exclude an artifactual association of these small RNAs with the 20S prosomal particle, in one case

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Fig. 8. Further purification of the Drosophila prosome by ion- exchange and hydrophobic chromatography. The fractions containing the prosome from a third run in a sucrose gradient in the presence of 10 mM NaCl were pooled and loaded onto (A) a 10- ml DEAE-Sephacel column (Pharmacia) equilibrated with 10 mM Tris (pH 7.5), l0 mM MgCl2, and l0 mM NaCl. The column was then washed with two volumes of the same buffer (minus sucrose) and eluted with a 30-ml gradient of l 0 mM to l M NaCl.

In each fraction the absorbance at 280 nm and the conductivity were measured. The peak containing the prosome (fractions 5- 9) was diluted with four volumes of l0 mM Tris (pH 7.5) and l0 mM MgC12 and loaded onto (B) a 5-ml-~0-aminodecylagarose column equilibrated with l0 mM Tris (pH 7.5), l0 mM MgC12, and 50 mM NaC1. After washing with the same buffer the elution was performed with a 20-ml gradient of 30 mM to 1 M NaC1

(Drosophila) we further analyzed this structure by chromatography. Figures 8 and 9 show the chromatography of the prosome on a DEAE-Sephacel column and subsequent chromatography on a hydrophobic (00-aminodecylagarose) column followed by a rerun in a sucrose gradient in the presence of 0.5 M NaC1. On DEAE-Sephacel equilibrated at pH 7.5, the prosome elutes at about 200 mM NaC1 (Fig.

8A). On the hydrophobic column, the 20S particle elutes at about 500 mM NaC1 (Fig. 8B). Some material is recovered in the flow-through of the hydrophobic column and contains essentially high-molecular-weight proteins (data not shown). After these chromatography steps, the Drosophila prosome re- sediments at 20S (Fig. 9A). The 280:260-nm ratio of the 20S absorbance peak indicates a ratio of proteins to RNA of about 95:5. The RNA present in each fraction of the gradient from 10 to 30S was purified, labeled in vitro with [32p]pCp, and analyzed in 7 M urea gels as described in Materials and Methods. Figure 9C shows that the same set of small RNAs (70-80 nucleotides long) still co-purify at 20S with the purified 20S particle, suggesting that they are tightly associated with this structure. T 1 fingerprint analysis of the RNA indicates that no major oligonucleotides have been lost during chromatography. The sedimentation of the prosome together with these small RNAs is not affected by an exten- sive treatment with pancreatic RNase (30 #g/ml for

1 h at 37~ in 5 mM MgC12).

Electron microscopic analysis indicates that the overall shape of the prosome is conserved following chromatography (Fig. 9D), but a decrease in the

Fig. 9. Protein and RNA constituent analysis of the Drosophila prosome purified by chromatography as described in Fig. 8. The fractions from the hydrophobic chromatography (Fig. 8B) containing the prosomal particle were further rerun on a sucrose gradient containing 0.5 M NaC1 as already described. A 280-nm (O--O) and 260-nm (D--D) absorbance profile of the sucrose gradient. B Coomassie-stained SDS polyacrylamide gel electrophoresis of the prosome (fractions 5, 6, and 7 pooled). C [32p]pCp-labeled RNA present in fractions 3, 4, 5, 7, 8, and 9 of the sucrose gradient. The gel is 7 M urea-8% acrylamide as above, nt, Nucleotides. D Electron microscope analysis of the chromatography-purified prosomal structure. Bar = 30 nm

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length of the cylinder and an increase of the diameter of the central dark zone of the round particle are observed.

Discussion

We have performed a comparative analysis of a particle called the prosome isolated from yeast, wheat, fly, chicken, rabbit, and human. In each case this particle sediments at about 20S and can be dis- tinguished from other cellular components by its conserved appearance as a hollow cylinder of 13- 14-nm diameter and 17-19-nm length and its composition of four stacked subunit rings. These conserved dimensions are close to those described in the cases of pea seedlings (Shelton et al. 1970), Xen- opus oocytes (Kleinschmidt et al. 1983), and duck cells (Schmidt et al. 1984).

In contrast to its ubiquitous shape, the 12-18 protein constituents of the prosome are not identical between species. However, from yeast to human, some polypeptides show homologous molecular weights and isoelectric points, suggesting they are possibly related. Thus, we may suggest that the proteins of the prosome are an example ofa polypeptide family that forms a conserved structure but has di- verged during evolution. In all the species we have analyzed, differing protein composition of the prosomes has been found. Therefore, we are as yet un- able to predict the protein stoichiometry in the prosome. It is possible that a multimer of even one individual protein can form a prosomal structure.

Antibodies specific to each of the different polypeptides of the prosome will help to resolve this.

We have described (Arrigo et al. 1985) that in Drosophila some of the protein constituents of the prosome are related to the low-molecular-weight heat-shock proteins (Arrigo 1980; Arrigo et al. 1980;

Arrigo and Ahmad-Zadeh 1981). Preliminary experiments indicate that the major 26-kd polypeptide present in the yeast prosomal preparation cross- reacts with an antibody raised against the low-molecular-weight heat-shock protein HSP26 (S.

Lindquist, personal communication). These obser- vations suggest that the similarity between the low- molecular-weight heat-shock proteins and some of the constituents of the prosome may be a general phenomenon.

We have found in each species analyzed small RNAs with electrophoretic mobilities close to tRNA co-purifying with the prosome; this observation agrees with analyses performed by others in chicken (Schmidt et al. 1984) and Drosophila (Schuld and Kloetzel 1985). However, other authors have reported in the cases of Xenopus oocyte nuclei (Kleinschmidt et al. 1983) and mammalian cells (Harmon et al. 1983) that a particle closely related

149 tO the prosome was purified free of RNA. We cannot rule out the possibility that nuclei of Xenopus may contain a population ofprosomes free of RNA, but these discrepancies could rather be due to either the ammonium sulfate precipitation (Kleinschmidt et al. 1983) or the hydroxylapatite chromatography (Harmon et al. 1983) steps used by these authors to purify the prosome. Because in our case these small RNAs still co-purify with the Drosophila prosome after two different types of chromatography (ion exchange and hydrophobic) we assume this association is not an artifact. How these small RNAs in- teract with the prosomal structure remains unknown, but judging from the results of Kleinschmidt et al.

(1983) and Harmon et al. (1983), they probably do not contribute to the overall shape of the particle.

Sequencing studies (Arrigo et al. 1985) and T I fingerprint analysis (this paper) indicate that most of these RNAs are different from the bulk of tRNAs.

Until reconstitution of functional particles can be achieved, the relevance of this putative association between RNA and particles will remain unknown.

It is perplexing to find such a highly organized structure so well conserved during evolution and not to have the remotest idea about its function.

Preliminary experiments performed with the prosome of Drosophila allow us to conclude that neither protein nor nucleotide kinase co-purifies with this structure, and it is similarly free from RNase activity. A hypothesis of a possible role of the prosome in the control of translation has been proposed (Shelton et al. 1970; Schmidt et al. 1984). It is in- teresting to find the prosome in the postribosomal fraction of two well-defined in vitro translational systems, i.e., in wheat germ and in rabbit reticulocyte lysate. Thus, specific anti-prosome antibodies will be useful in testing whether or not this particle plays a role in the control of translation. It is in- triguing to note that recently Castagno et al. (I 986) have detected a 5'-tRNA maturase activity co-purifying with a similar particle isolated from Xenopus oocyte nuclei. Finally, we cannot exclude the pos- ibility that the prosome is a small viroid-like particle parasitizing most of the eukaryotes for more than a billion years.

Acknowledgments. We thank W. Welch and J. Lamb for a crit- ical reading of the manuscript, F. KeppeI-Ballivet for yeast cultures, E. Boy de la Tour for his help in the electron microscopy analysis, and F. Mulhauser for excellent technical assistance. This research was supported by the Swiss National Science Founda- tion.

References

Arrigo A-P (1980) Investigation of the function of the heat shock proteins in Drosophila melanogaster tissue culture cells.

Mol Gen Genet 178:517-524

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Received June 17, 1986/Revised December 1, 1986