The intracellular distribution of basic proteins in the Chinese hamster ovary cell

Q 1968 by

238

Experimental

THE INTRACELLULAR DISTRIBUTION IN THE CHINESE HAMSTER G. R. SHEPHERD

Academic Press Inc.

Cell Research 49, 238-250 (1968)

OF BASIC PROTEINS OVARY CELL

and B. J. NOLAND

Los Alamos Scientijic Laboratory, University of California, Los Alamos, N. Mex., 87544, USA

Received March 8, 1967

has been suggested that the histones, a family of basic proteins, may function as direct regulators or intermediate moderators of gene expression [13, 16, 291. Their close proximity to nuclear DNA and their ability to influence both in vivo and in vitro nucleic acid polymerase reactions tend to support this role for histones [l, 141. C onsiderable efrort has been expended investigating histones isolated from whole tissue, nuclei, chromatin and washed uucleoprotein (see reviews [4, 5, 231). It is becoming increasingly obvious, however, that basic proteins are not found exclusively in nuclear chromatin and that histones or histone-like basic proteins are widely distributed throughout the cell [3, 7, 18, 19, 34, 351 and may migrate between the cytoplasm and nucleus [25]. The relationship between extranuclear basic proteins and “authentic” nuclear histones must be studied and clarified before conclusions may be drawm concerning their respective roles. It would be especially valuable if such comparisons were made using a single biological source. The present report, part of a larger investigative effort, deals with the isolation and comparison of basic proteins obtained from a variety of intracellular loci within the cultured Chinese hamster ovary cell. IT

MATERIALS

AND

METHODS

Cell culture.-Chinese hamster ovary (CHO) cells exhibiting a modal chromosomal number of 22 [30] were grown at 37°C in suspension culture in F-10 medium [li] supplemented with IO per cent calf and 5 per cent fetal calf sera. All growth media contained 100 units/ml of penicillin and 100 yg/ml of streptomycin. Cells used in these experiments were derived from a sub-clone (CHO-10) with a generation time in fluid culture of 16 h and exponential growth between 5 x lo4 and 5 x lo5 cells/ml. Cell densities were determined using electronic particle counting methods [17, 321. Amino acid analyses.-Protein preparations were hydrolyzed for 22 h in 6 N HCl at 110°C in evacuated and sealed tubes. Amino acid analyses were performed on a Beckman Spinco automatic analyzer (Model 120B) equipped with computer-compatible digital punched tape data output [9]. Results are listed in Table 1. Experimental

Cell Research 49

Infrcrcellular

distribution

239

of basic proteins

Electrophoretic analyses.-Polyacrylamide gel disc electrophoretic analyses were performed using a previously described method [26]. Alizarin Black replaced the less stable Amido Black 1OB dye in this system. Stained gels were scanned with a Joyce-Loebl Chromoscan photodensitometer equipped with a 0.2 x 2-mm slit, O-2.0 optical density wedge and a 1: 10 carriage transport reduction gear. T;ZI~LF; 1. Amino

crcid compositions

of brrsic protein

preptrrcrtions.

Chinese hamster ovnry (ymole per ceni) Amino

acid

Lysine Histidine hrginine Aspartic Threonim? Serina’ Glutamic Proline Glycine Alanine Half cystinc Valinc Melhionine Isoleucinc Leucine Tyrosine Phenylalanine

Micro-

Ribo-

Lpso-

Nuclear

Chromatin

Nucleolar

somal

somal

somal

14.3

13.3

13.ti

12.4

12.1

1 1.0

12.4

1.8

2.1

2.2

2.1

2.1

2.2

2.0

9.2

8.7

9.2

8.3

7.9

9.2

7.7

4.9

5.6

5.7

6.5

8.1

7.4

7.4

5.8

5.5

5.7

5.8

5.2

5.1

5.6

5.1

5.2

5.7

5.8

5.3

5.0

6.i

8.4

9.5

9.1

9.7

10.1

9.0

10.2

Calf thymus

4.8

5.2

5.2

5.1

5.7

5.2

5.4

9.3

9.1

8.8

9.4

9.0

7.8

8.6

13.8

12.1

11.5

11.3

0

0

0

0

8.7

9.1

9.5

0

0

0

5.8

6.3

6.5

6.6

7.2

7.3

6.4

0.7

0.9

0.3

0.1

1.0

0.9

1 .o

3.9

4.4

4.5

4.8

4.9

4.9

4.5

7.9

7.6

7.7

8.1

7.3

7.4

7.3

2.6

2.4

2.4

1.7

2.0

2.5

2.5

1.6

2.1

2.1

2.4

3.3

2.9

2.8

Samples hydrolyzed in 6 N HCI, llO”C, 22 h. a Serine and threonine corrected for loss. Whole cell extraction.-Saline-washed cells were extracted at 2-5°C in a Teflonglass homogenizer with 10 vol of each of the solutions indicated in Table 2. Each homogenate was allowed to extract for IO min, after which it was clarified by centrifugation at 30,500 g for 10 min. Each pellet was reextracted as before with the same solution before proceeding to the next step. The supernatant from each extraction was treated with 5 vol of cold acetone and permitted to stand in the cold overnight. Aliquots of each superantant before addition of acetone were brought to 2O”C, and their pH values were determined. Acetone precipitates were collected by centrifugation, washed with a I:1 acetone-ethanol mixture and dissolved in a minimal volume of distilled water. The resulting solution was clarified by centrifugation at 105,000 g for 10 min, and the supernatant was lyophilized. Recoveries are presented in Table 2. Experimental

Cell Reseurch 49

G. R. Shepherd

240

nnd B. J. Noland

Whole cell fractionation.-Washed CHO cells were homogenized extensively in 1.0 mM PO,-0.5 mM MgCl,, pH 7.2, hypotonic medium, and the homogenate was separated into particulate fractions by differential centrifugation. Each fraction was washed with the buffer and sequentially extracted with dilute sulfuric acid solutions of pH values 3.0, 2.0, 1.0 and 0.5. The homogenate was clarified at each T.\BLE

2. Extraction

schedule

for CHO

cells.

pHa

Yield (W

Basic proteinb

Saline citrate Distilled H,O H&O,, pH 4.0 H,SO,, pH 4.0

6.70 6.60

72.250 40.750 0.294 3.978

Segative Faint Faint Faint

H,SO,, H,SO,, H,SO,, H,SO,,

pH pH pH pH

3.0 3.0 2.5 2.5

4.73 3.55 2.80 2.75

0.186 0.786 0.519 Trace

Faint Faint Positive Positive

9 10 11 12

H,SO,, H&O,, H,SO,, H,SO,,

pH pH pH pH

2.0 2.0 1.5 1.5

2.35 2.22 1.73 1.63

0.384 0.100 0.290 0.126

Positive Positive Positive Positive

13 14 15 16

H,SO,, H,SO,, H,SO,, H,SO,,

pH pH pH pH

1.0 1.0 0..5 0.5

1.23 1.23 0.63 0.62

0.214 Trace Trace Trace

Positive Positive Positive Positive

17 18

W’ NaOH,

0.01 N

Trace Trace

Negative Xegative

NO.

a Temperature b As evidenced

Solution

of extraction 2%5°C; temperature of pH measurements by presence or absence of banding on polyacrylamide

20°C. gel disc electrophoresis.

step by centrifugation at 105,000 g for 5 min. The sequential extracts from individual fractions were pooled and treated with 5 vol of cold acetone. After standing overnight the precipitates were harvested by low-speed centrifugation, washed once with cold 1 :l acetone-ethanol and dissolved in a minimal volume of water. The solutions were clarified as before, and the supernatants were freeze-dried. This procedure was used for all succeeding extractions. Electrophoretic banding patterns for each fraction are seen in Fig. 1. Nuclei.-Nuclei were isolated from CHO cells by hypotonic shock, by the method of Allfrey et al. [2], by modifications of a detergent procedure [15] and by the method of Chaveau et al. [6]. Of these, the method of Chaveau, the method of Allfrey and the method of hypotonic shock gave intact nuclei. Basic proteins isolated from nuclei Experimental

Cell Research 49

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of busic proteins

241

prepared by each of these methods showed identical gel banding patterns. The following method of hypotonic shock was chosen for routine nuclear preparation on the basis of its speed, simplicity and reliability. Washed CHO cells were suspended with gentle agitation in 20 vol of cold 1.0 mM PO,-0.5 mM MgCl, buffer adjusted to pH 7.1. The suspension was allowed to stand for 15 min, after which it was gently homogenized with 15 strokes of a Dounce “A” hand homogenizer. Nuclei were collected by centrifugation at 500 g for 10 min, resuspended in 20 vol of fresh buffer with 3 strokes of the homogenizer and collected as before. The pelleted nuclei were free of visible cytoplasmic contamination when examined by phase contrast microscopy. Basic proteins were extracted from nuclei and subjected to electrophoretic analysis (Fig. 2). ~Vuclear chromatin.-Nuclei were extensively homogenized in a micro blender with 20 volumes of 0.14 $1 NaCl-0.005 M MgCl,-0.02 111Tris Cl, pH 7.5, yielding ribo-

Fig. 1..--Electrophoretic banding patterns for cell particulate fractions: A, nuclear, 500 g, 15 min; B, large particulate fraction, 27,000 g, 15 min; C, small particulate fraction, 105,000 g, 120 min; and II, soluble fraction.

A

Experimmtul

Cdl Resectrch 49

242

G. R. Shepherd

and 13. J. Xoland

Fig. %.-Electrophoretic banding patterns of basic proteins derived from A, calf thymus hislone; B, whole CHO cell basic protein; C, CHO nuclei; D, CHO nucleoli; E, CHO washed chromatin; F, CHO lysosomes; C, CHO microsomes; and H, CHO ribosomes.

some-free nuclei or washed chromatin [33]. The homogenate was clarified by centrifugation at 25,000 g for 15 min. Basic proteins were prepared from the pellet as described and subjected to electrophoretic analysis with the results illustrated in Fig. 2. iVucZeoZi.-Nucleoli were isolated from nuclei by the methods of Desjardins et al. [IO]. In view of the uncertainty concerning the state and role of nucleolar chromatin, no attempt was made to remove associated residual chromatin threads. Basic proteins were prepared as described and subjected to electrophoretic analysis (Fig. 2). Microsomes and ribosomes.-Microsomes were isolated by fractional centrifugation from the cytoplasmic brei of nuclear preparations. Ribosomes were obtained by 0.5 per cent deoxycholate treatment of microsomes, followed by centrifugation at 105,000 g for 2 h. Basic proteins were prepared from microsomes and ribosomes as previously described. Electrophoretic banding patterns for representative preparations are illustrated in Fig. 2. Mitochondria.-Mitochondria were isolated from CHO cells by the method of Hawtrey and Silk [12], purified by centrifugation through a 5525 per cent sucrose gradient and extracted as previously described. Considerable material was recovered which had a neutral composite amino acid composition and which was only partially Experimental

Cell Research 49

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of basic proteins

213

soluble in water or gel buffer. Mitochondria from Neurospora crassa were the generous gift of Dr K. Munkres. Beef heart mitochondria were prepared by the method of Criddle et al. [8]. Lysosomes.-CHO cells were disrupted by high-speed homogenization in 0.45 II2 sucrose at pH 7.0 containing 6.8 m&Z EDTA [27]. The homogenate was separated by differential centrifugation into 7 fractions. Each fraction was washed once with buffer, treated with 0.1 per cent Triton X-100 and assayed for acid and alkaline phosphatase [31] and succinoxidase [28] activities. The results are displayed in Fig. 3. Basic proteins were isolated from fraction 5 and subjected to gel electrophoresis (Fig. 2). Nuclear ribosomes.-Repeated attempts to isolate ribosomes from nuclear homogenates as described by Wang [33] were unable to provide a particulate fraction uncontaminated with amorphous material and chromatin fragments.

RESULTS Whole

cell extmction

hImray has demonstrated that histone fractions may be selectively removed from calf thymus nucleoprotein by hydrogen ion titration [‘La]. A similar extraction was performed with CHO cells according to the schedule in Table 2. The pH values and yields for each extract are also recorded in Table 2. Large amounts of material were obtained by extraction with saline, water and dilute acid solutions, but it was not until an extract value of pH 2.8 was reached that appreciable quantities of basic proteins were recovered, as evidenced by the presence or absence of banding when extracts were subjected to electrophoresis. Extraction of basic proteins continued until an extract value of pH 0.63 was reached. From these data we may conclude that the limits of CHO nucleoprotein dissociation extend from pH 2.8-0.6. All succeeding extraction procedures were designed to comply with these limits. TVhole cell fractionation

Basic proteins obtained from particulate fractions prepared by differential centrifugation demonstrated complex patterns with considerable quantitative and qualitative variation in individual bands among the fractions (Fig. l), indicating that a partition of basic proteins had occurred during the fractionation procedure. 155th the exception of a single faint forerunner band derived from the 105,000 g-supernatant, acid-soluble basic proteins of the CHO cell were found to be associated with particulate fractions or to form sedimentable complexes upon disruption of the cell. Experimenfnl

Cell Research 49

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G. R. Shepherd and B. J. Noland

Nuclei Basic proteins obtained from isolated nuclei exhibited reproducible banding patterns (Fig. 2) which were notably less complex than whole cell extract patterns and which could be distinguished by the quantitative and qualitative distribution of the individual bands. Their banding patterns were remarkably constant from one preparation to the next, as illustrated by the densitometer tracings of gel patterns of 4 different preparations seen in Fig. 4.

-GEL

Figs 3 and 5.

LENGTH

Fig. 4.

Fig. 3.-Distribution of A, alkaline phosphatase; B, acid phosphatase; and C, succinoxidase among cell particulate and soluble fractions. Fractions were separated by differential centrifugation as follows: (1) 650 9, 10 min; (2) 4080 9, 10 min; (3) 16,300 g, 10 min; (4) 27,000 g, 10 min; (5) 78,400 g, 10 min; (6) 78,400 g, 60 min; and (7) final supernatant. Abscissa: Fraction number; ordinate: y0 of total enzyme activity. Fig. 4.-Densitometer tracings of electrophoretic banding patterns of four individual CHO nuclear basic protein preparations. Fig. 5.-Densitometer tracings of electrophoretic banding patterns of A, calf thymus histone; and B, CHO nuclear basic protein. Experimental

Cell Research 49

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distribution

of basic proteins

215

The similarities between the gel banding patterns of calf thymus histone and CHO nuclear basic protein may be seen by comparing their densitometer tracings presented in Fig. 5. The quantitative validity of such comparisons was demonstrated in the following manner. A series of polyacrylamide gels were loaded each with 30 ,ul of solutions containing from 25-125 pug of calf thpmus histone. The gels were subjected to parallel electrophoresis, stained overnight and electrically destained. Each gel was scanned in a photodensitometer, and the portion indicated in Fig. 6A was traced with a planimeter. The areas were plotted against concentration as in Fig. 6B. It may be seen that total area is a linear function of protein concentration within the range tested. Individual peak areas were also found to demonstrate a linear response to protein concentration with each slope dependent upon the ability of the individual protein fraction to bind alizarin dye as well as upon the spectral characteristics of each protein-dye complex. Xrrclear chromatin Vigorous homogenization of isolated nuclei with saline-Tris solution to produce chromatin reduced the number of major electrophoretic bands from 16-12 (Fig. 2) and decreased the faint fine structure originally observed near the origin. It could not be readily determined if the loss of major bands was the result of removal of contaminating material or was effected by denaturation of the DNP complex with a concomitant extraction of less tightly bound proteins. It is interesting to note that calf thymus histone,

+-AREA

TRACED-

4

Fig. G.-Effect of protein load on electrophoretic pattern band intensity: A, densitometer tracing indicating area to be measured; B, plot of protein load versus measured area of curve above base line. Experimental

Cell Research 49

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G. R. Shepherd and B. J. Noland

prepared by acid extraction of saline-washed thymus tissue, produced a banding pattern similar to those of the basic proteins of the CHO cell but intermediate in complexity and banding intensities between CHO nuclear and CHO washed chromatin preparations.

Electrophoretic patterns obtained from nucleolar basic proteins were similar to those of whole nuclei and identical to those obtained for washed chromatin (Fig. 2). No unique bands attributable to the nucleolar bodies proper were observed in these preparations. The relative intensities of individual bands were similar in nucleolar and chromatin patterns, indicating a similar distribution of basic proteins in nuclear chromatin and nucleoli and nucleolar chromatin. The lack of significant complex banding near the origin indicates only minor contamination of these preparations vvith cytoplasmic material. Microsomes and ribosomes No differences were observed between the banding patterns of microsomal and ribosomal basic proteins (Fig. 2), indicating that deoxycholate treatment of the microsome does not remove basic protein not also present in the ribosome proper. Visual inspection of freshly prepared gels revealed the presence of 33 distinct bands, 11 of which were located near the origin. Variations in staining intensity among individual bands were considerably less than that exhibited by nuclear and chromatin preparations, suggesting that microsomal and ribosomal particles contain a complement of basic proteins both more complex and more homogeneous in distribution than nuclear chromatin. p--D-

--i---c-

--A-

--i-B

ORIGIN-

: Fig. 7.-Densitometer tracings CHO ribosomal basic proteins. Experimental

Cell Heseareh 49

of electrophoretic

banding

patterns

for A, CHO nuclear,

and B,

Intracellular

distribution

of basic proteins

247

Densitometer tracings of stained gels representing CHO nuclear and ribosomal basic protein banding patterns are presented in Fig. 7. For purposes of comparison, the tracings have been divided into regions A-D with respect to major band groups and their distances from the origin. Ribosomal region ,4 contained a complex of at least 11 tine bands, while washed chromatin contained only one band in this region. Ribosomal region B contained respectively a pair and a triad of bands at the positions occupied in nuclear preparations by three major bands. Ribosomal region C contained two bands which were not observed in nuclear preparations. In region D two faint nuclear forerunner bands were present which did not appear in the ribosomal preparation. Mitochondria No bands were observed when acid-soluble mitochondrial proteins were subjected to electrophoretic analysis. Similar results were obtained with mitochondria isolated from beef heart and from Neurospora crassa. In view of the extremely low reported mitochondrial DNA4 content [20], it was to be expected that basic proteins associated with mitochondrial DNA would be present in extremely low quantities and might indeed be present as an insoluble complex with more acidic proteins. L ysosotnes Basic proteins have been demonstrated in lysosomes isolated from rabbit polymorphonuclear leukocytes [34, 351. Cytochemical evidence has been obtained for the presence of lysosome-like particles in cultured mammalian cells [21, 221. Differential centrifugation of a CHO cell homogenate produced a series of seven fractions. Of these, fraction 5 exhibited maximal acid and alkaline phosphatase and succinoxidase activities, indicating a mixture of lysosomal and mitochondrial particles. Basic proteins isolated from fraction 5 demonstrated a banding pattern in which region A major bands resembled those of ribosomal protein, while region B simulated nucleolar preparations. In addition, the lysosomal pattern contained three bands in region C which were present as major components in whole cell preparations, were observed as possible trace components in ribosomal preparations and were absent in nuclear preparations. The outstanding ribosomal region A line structure and region 13 doublet-triplet bands were absent in lysosomal patterns, attesting to the absence of microsomal contamination, while the absence of nuclear chromatin region A doublet attested to the absence of contaminating chromatin material. 17 - 681804

Experimental

Cell Research 49

248

G. R. Shepherd and B. J. Noland DISCUSSION

Nuclear DNA of higher organisms occurs complexed with basic proteins, the histones, as deoxyribonucleoproteins. The close association of histones and DNA in the nucleus suggests a functional relationship which may involve the structure, or moderation of the transcription, of nuclear DNA. Since histones are predominantly nuclear in origin and since basic proteins are historically considered to be confined to complexes with nucleic acids, histones have come to be defined in terms of these characteristics. Recent investigations have shown that basic proteins resembling nuclear histones may be found in extranuclear loci which contain little or no DNA. Lindsay [19] has demonstrated a striking resemblance between the starch gel electrophoretic patterns of basic proteins isolated from chromosomes (or chromatin) and ribosomes of chicken liver. Others have observed basic proteins in nucleoli [3, lo], microsomes and ribosomes [7, 181 and lysosomal particles [34, 353. We have shown that with a possible single exception the acid-soluble basic proteins of CHO cells are associated with particulate material or form sedimentable complexes upon disruption of the cell. Investigation of purified organelles demonstrated basic proteins in the nucleus, nucleolus, chromatin, cytoplasmic microsomes and ribosomes and the lysosomes of the CHO cell. be Each band observed in a whole cell extract electrophoretic pattern could accounted for by a summation of the bands of the organelle patterns. Basic proteins from whole cell extracts produced complex electrophoretic patterns containing 30 or more components. Basic proteins from intact nuclei produced distinct but less complex patterns, while the patterns of nucleolar and chromatin basic proteins mere identical and resembled those The similarity between proteins from these of whole nuclear preparations. essentially nuclear sites suggests a common origin, deoxyribonucleohistone or chromatin. Basic proteins obtained from lysosomal particles produced electrophoretic patterns similar to those of nuclear protein in gel regions A and B and contained two unique bands in region C. Microsomal and ribosomal basic protein patterns were identical and were readily distinguished from nuclear and lysosomal patterns. A comparison of parallel photodensitometer tracings of nuclear and ribosomal patterns revealed several points of similarity and a host of dissimilarities. It should be noted that while the majority of bands demonstrated by this method are comprised of basic proteins the inclusion of acidic proteins, either free or complexed with basic proteins, may not be excluded without Experimental

Cell Research 49

Intracellular

distribution

of basic proteins

249

further study. While the question of identity of individual bands must be answered by exhaustive composition and sequence analysis, a comparison of the electrophoretic patterns of organelle basic proteins suggests that nuclear sites and cytoplasmic lysosomes share the same main basic protein complement, with each species occurring in a proportion characteristic of the site. Cytoplasmic ribosomes not only share certain of these but also appear to contain their own unique complement of basic proteins. These results suggest that histones are the nuclear members of a family of basic proteins \videly distributed within the cell. Each of the basic protein-containing sites investigated is related in some degree to the mechanisms of genetic information transfer; they are directly involved in the replication and transcription of DNA (nuclei, nucleoli, chromatin) and in the transcription of DNA to protein (ribosomes) or represent temporary components of the mitotic apparatus (lysosomes). Thus the postulated participation of basic proteins in genetic information transfer may not be ruled out on the basis of their intracellular distribution. It is entirely possible, ho\\,ever, that the metabolism and function of otherwise identical basic proteins are to some degree site specific. It is also of interest to speculate that sharing of a given protein species between two or more sites might well lead to the possibility of equivalence and interchange of that species between sites [25]. SUMMARY

Ilasic proteins were obtained by acid extraction from whole cells and from nuclei, nucleoli, chromatin, cytoplasmic ribosomes and lysosomes of fluid cultured Chinese hamster ovary (CHO) cells. The limits of nucleoprotein dissociation ranged from pH 2.8-0.6. Basic proteins isolated from whole cells demonstrated complex electrophoretic patterns containing approximately 30 components. Basic proteins from nuclei demonstrated less complex banding patterns, while the patterns from nucleoli and chromatin qualitatively resembled those from nuclei. Lysosomal basic protein patterns resembled those of nuclei with three additional bands which were present in whole cell preparations, were observed as trace components in ribosomal preparations and were absent in nuclear basic proteins. Ribosomal basic proteins demonstrated a complex banding pattern which appeared both to share certain fractions with nuclear and lysosomal fractions and to contain a number of unique components. A comparison of relative band staining intensities among the preparations, both visually and by means of a photodensitometer, suggested for the comExperimental

Cell Research 49

250

G. R. Shepherd and B. J. Noland

mon bands a distribution of a mixture of basic proteins among intracellular sites, the proportions of which mixture varied according to the individual locus. This work was performed under the auspices of the US Atomic Energy Commission. The authors would like to express their appreciation to C. N. Roberts for her aid in performing amino acid analyses and to Dr L. R. Gurley for his suggestions and support. REFERENCES

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Experimental

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1, 175 (1963).