Subribosomal particles of HeLa cells

Printed in Sweden Copyright Q 1974 by Academic Press, Inc. All rights of reproduction in any form reserved

Experimental Cell Research 87 (1974) 253-2.58

SUBRIBOSOMAL

PARTICLES

OF HELA CELLS

M. SMULSON Department of Biochemistry, Georgetown University,

Schools of Medicine and Dentistry, Washington, D.C. 20007, USA

SUMMARY Well defined ring-shaped and rectangular particles are described as intracellular components of post-ribosomal cytosol of HeLa cells. Their morphology might be identical with particles recently isolated from plasma cell tumors and independently from the culture medium of malignant cells. The HeLa particles exist throughout post-ribosomal extracts from 30-10s; however, the highest concentration and best resolution exists at approx. 14-17s. Ultrastructure by electron microscopy indicates aggregation of the particles at higher sedimentation values. The possible relationship of high molecular weight forms of protein synthesis translational enzymes with both the intra- and extracellular particles has been explored.

A recent report by Narayan & Rounds [l] has described the occurrence of ring-shaped particles containing DNA, RNA and protein with distinct morphology in the extracellular tissue culture medium (serum-containing or serum-free) of only rapidly dividing cultures of human adenocarcinoma, HeLa and KB cells. The biological function of these particles has not been ascertained; they were identified by the above workers’ attempt to purify growth modifying factor activity associated with medium. In this present communication we wish to report ultrastructural studies characterizing post-ribosomal particles from the cytosol of HeLa cells which closely resemble the particles isolated from culture medium by Narayan & Rounds. The possible function of these particles in protein synthesis has been explored. MATERIALS

AND METHODS

Conditions for cell growth have been described earlier [2].

Postribosomal cell extract Postmitochondrial cell extract was prepared as described previously [3, 41. The extract was layered on 5-20 % linear sucrose gradients in reticulocvte standard buffer (10 mM ‘iris, HCI, pH 7.4,
Assay of elongation factor 2 The diphtheria toxin catalyzed ADP-ribosylation of elongation factor 2 has been described in detail earlier [2, 31.

Electron microscopy Samples of postribosomal cell extracts were prepared for electron microscopy as follows: one droplet of sample was deposited on the electron microscope grid. After approx. 2 min, excess of liquid was removed by absorption to filter paper, and the remaining material was negatively stained for 2 min either in a 2 % solution of potassium phosphotungstate (pH 6.2) or in a 2 % uranyl acetate solution. In some cases (samples of very low protein concentration) the grid was covered with a thin layer of bovine serum albumin before deposition of the sample. Electron micrographs were taken at an initial magnification of 60 000 with a Hitachi HU-1 I-E Electron Microscope. Exptl

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M. Smulson

Fig. I. Abscissa: no. of drops; ordinate: (left) transferase II/200 yl (dpm x 1O-s); O-30: S values (0-o); (right) UdR incorp./lOO ,~l (cpm x 10-3) (a--n). Postribosomal distribution of EF-2 and non-ribosomal RNA after Nonidet-P40 lysis of HeLa cells. Two cultures of HeLa cells (4 x lo5 cells/ml) were incubated for 4.5 h at 37°C in the presence of actinomycin D (0.05 pg/ml). Non-ribosomal RNA in one of the cultures was labeled with 3H-uridine (0.04 PM, 1 &i/ml). Post-mitochondrial extracts were prepared after lysis of the cells by Nonidet-P40, and samples of the two extracts were analyzed on separate 5-20 % sucrose gradients (36 ml), A third gradient contained ‘Glabeled RNA standards. The two sample-containing gradients were correlated with the RNA-standard-gradient. The electron micrographs of sub-ribosomal particles shown at the bottom of the figure were prepared from fractions corresponding to the position of the pictures.

RESULTS Distribution of subribosomal particles in postribosomal cell extracts In the experiment described in fig. 1 HeLa cells were gently lysed by treatment with Nonidet-P40 [4], post-mitochondrial extract was prepared and this extract was centrifuged through a 5-20 Y0 sucrose gradient as described in Materials and Methods in order to display post-ribosomal material of 30s and less. Well characterized radioactive HeLa Exptl Cell Res 87 (1974)

ribosomal RNA (fig. 1) was utilized to assign sedimentation values. Identical results were obtained when cells were disrupted by hypotonic shock [3]. This array of cellular material was initially investigated in order to better characterize the size of cellular particulate material, the soluble pellet [3, 61 which we have reported contains activity for a number of aminoacyl-tRNA synthetases, as well as elongation factors 1 and 2. Electron micrographs of subribosomal particles are inserted in fig. 1 approximately be-

HeLa cell particles

255

Fig. 2. (a, b) Representative views of rectangular and elongated particles sedimenting at 25-30s. At least one of the large particles seems to be composed of several of the smaller ones; (c) uranyl acetate stained ring-shaped particles that appear to be composed of subunits. (d) ring-shaped particle with two ‘end-pieces’. Uranyl acetate stain. x 180 000. (Scale 250 A).

low the fractions from the gradients from which they were obtained. These particles were found throughout the gradient from approx. 8s to 3OS, however, the highest concentration was localized in the 14-17s portion of post-ribosomal material. Two very characteristic particles can be recognized in all the postribosomal extracts studied in our laboratory, regardless of the means of preparation: a 105-l 15 x 165 A rectangular particle (fig. 1, fig. 2b), and ring-shaped particles with diameters ranging from 105115 A (fig. 2c, fig. 3). The ring-shaped particles seem identical to the extracellular particles described by Narayan & Rounds [l], although it is of interest to note that the rectangular particles are also evident in their

electron micrographs. Shelton et al. also have described intracellular particles from plasma cell tumors with similar morphology and sedimentation values as those described above [7]. At higher S value ranges of post-ribosomal extracts, we found that the particles were more scarcely distributed, and at lower S values large amounts of amorphous material made it difficult to distinguish the particles, although they were present even in fractions corresponding to 10 S. Morphology of subribosomal particles At higher S values of post-ribosomal cytosol derived from HeLa cells, a new particle form is apparent (fig. 2a). This particle is larger Exptl Cell Res 87 (1974)

256 M. Smulson

Fig. 3. Large scale magnification of ring-shaped and rectangular particles. One of the ring-shaped particles

may have collapsed. x 480 000. (Scale 250 A).

and more elongated than the above mentioned (approx. 85 x 500 A) and seemsin some cases to be composed of several of the rectangular particles (fig. 2b). Fig. 2c and d show the small particles stained with uranyl acetate. The ring-shaped particles are clearly visible and seem to be the dominating species under these staining conditions. It is of interest that this species is most evident in the extracellular particles of Narayan & Rounds, who also stained with uranyl acetate. Some of the particles (fig. 2b, c) appear to be composed of 3-4 subunits. Exptl Cell Res 87 (1974)

Large scale magnification of both the ring-shaped and the rectangular particles is shown in fig. 3. The subunit structure of the ring-shaped particles that was noted in fig. 2 is not so apparent at high magnification. Some of the particles appear to have a disc rather than a ring-shape; since the size of the disc-shaped particles is smaller than that of the ring-shaped ones, they might, however, have been formed by collapse of the ring-shaped particles. The rectangular particles can be visualized as a stack composed of 3-4 ring-shaped particles, or alternatively,

HeLa cell particles a ring-shaped particle to which is attached two ‘end-pieces’. Each of the ‘end-pieces’ has the shape of a bar (approx. 17 x 110 A). The latter type of rectangular particle could conceivably be formed from the other type, if the ring in the middle of the stack was turned around; if this actually is the case, they probably represent a degradation product of the stacked-type particles. It is of interest that the particles described here seem to bear some resemblance to the structure of a complex of aminoacyl-tRNA synthetases described by Vennegoor & Bloemendal [S]. The complex described by these authors were prepared in a way very similar to the soluble pellet which we have described 13, 5, 61. Size heterogenenity of translation enzymes in post-ribosomal cell cytosol The data in fig. 1 shows the distribution of elongation factor 2, as assessed by the highly specific diphtheria toxin adenosine diphosphoribosylation reaction [3], in postribosoma1 HeLa cell extracts. The data confirms our previous observations [5, 61 that this enzyme exists as an aggregated form at sedimentation values from lo-17S, in addition to the free unassociated species at 5s. Elsewhere we have shown that the 10-17s range of post-ribosomal cytosol possesses aggregated forms of elongation factor 1 and various aminoacyl-tRNA synthetases [5, 61. The highest concentration and best resolution of rectangular particles was also found in this size range, however, it should be emphasized that until further purification is completed, there is no evidence that the morphology and enzyme activities are necessarily related in the intact cell. It can, however, be firmly stated that the particles isolated from medium of cells described by Narayan & Rounds [l] also exist in, and could be derived from the cytosol of intact cells.

251

We have also isolated a pellet from HeLa cell culture medium using the procedures suggested by Narayan & Rounds [l]. Our preparation contained particles with identical morphology as those described by fig. 2b and c, and with those described by the above workers. It is of interest that this medium pellet showed definite activity for elongation factor 2 (1.6 x lo3 dpm incorporation of ADP-ribose from NAD only in the presence of diphtheria toxin), and activity for the aminoacyl-tRNA synthetases for leu, ileu, ala, ser, and gly. Again, any inference of a relationship between the coincidence with morphology and activity must await further purification of the enzyme activities and particles. Finally, the data in fig. 1 indicates that when cells are incubated with actinomycin D under conditions where rRNA synthesis is inhibited, uridine is incorporated into RNA which sediments heterogeneously in the range where rectangular and circular particles are found. Narayan & Rounds have reported that their particles, which seem identical to the particles we visualize from intracellular material, contain RNA, as well as DNA and protein. DISCUSSION The particles analysed in HeLa cytoplasm seem identical to the extracellular particles isolated by Narayan & Rounds in the purification of growth modifying factor from culture medium [l] and the particles described by Shelton et al. [7] derived from cytosol of plasma cell tumors. The latter authors found correspondence between the highest concentration of particles and a high molecular weight form of elongation factor 1. The basic unit of all these particles is a ringshaped particle since this type was seen alone or in combination with two or three other particles of the same appearance, thus formExptl Cell Res 87 (1974)

258 M. Smulson ing the rectangular form. The largest concentration of these particles were found at 14-17s. It is rather puzzling, however, that the particles were heterogeneously distributed throughout the 10-30s range of the sucrose gradients (figs 1, 2). Subcellular material with well characterized morphology should be expected to sediment at specific S values. One possible explanation for the heterogeneity of the particle distribution might be that the observed particles really are fragments of one or more larger complexes. Indeed, the electron micrographs in fig. 2a of particles sedimenting at 25-308 show considerable aggregation. Such a large complex might be relatively fragile, and disassociate to the smaller units during gradient centrifugation. Perhaps related to this problem of size heterogeneity might be the recent preliminary report by Weissbach [9] that a huge lipid rich complex of many translational enzymes has been isolated. Differing amounts or distributions of lipids could conceivably account for the heterogeneous densities of particles which all seem identical in basic morphology. How these post-ribosomal particles, the extracellular particles of Narayan [I], the particles described by Shelton [7] and perhaps the morphology for the aminoacyltRNA synthetase complex described by Vennegoor & Bloemendal [8] relate or do not relate to the enzyme activities for elongation factors 1 and 2 and synthetases is far from clear at this time. An anticipated further purification of all the activities hopefully will begin to answer these questions. Even then, non-specific association of many differing enzymes with cell structures cannot be ignored. However, our findings as described above of elongation 2 activity and

Exptl Cell Res 87 (1974)

aminoacyl-tRNA synthetase activity with the extracellular particle with identical morphology to these other forms is promising. We have previously shown that the enzyme aggregate, the soluble pellet [3, 61, possibly has an important cellular function, since elongation factor 2 association with this subcellular fraction varied according to the rate of protein synthesis of the intact HeLa cell [3]. Latter, we showed that other activities required for protein synthesis seemed also aggregated with this cell fraction [5, 61. One could envision that the speed and efficiency of cellular protein synthesis might be higher if all the enzymes required for translation were complexed together. A distinct particle nature, such as those described above, for such a complex is possible, although at this stage of development such a conclusion would be an overinterpretation of the existing evidence. This investigation was supported by a Vincent Lombardi Memorial Grant for Cancer Research from the American Cancer Society, and a USPHS grant (CA11950).

REFERENCES 1. Narayan, K S & Rounds, D E, Nature new biol 243 (1973) 146. 2. Smulson, M E & Rideau, C, J biol them 235 (1970) 5350. 3. Henriksen, 0 & Smulson, M, Arch biochem biophys 150 (1972) 175. 4. Borun, T W, Scharff, M D & Robbins, E, Biochim biophys acta 149 (1967) 302. 5. Smulson, M & Gazzoli, M. Submitted for publication. 6. Henriksen, 0 & Smulson, M, Biochim biophys res commun 49 (1972) 1047. 7. Shelton, E, Kuff, E L, Maxwell, E S & Harrington. J T. J cell biol 45 (1970) 1. 8. Vennegdor, C & Bloemendal, H, Eur j biochem 26 (1972) 462. 9. Weissbach, H, Nature new biol 244 (1973) 97. Received January 9, 1974