Stomatocytic or discoidal erythrocyte ghosts containing only spectrin

BIOCHEMICAL

Vol. 168, No. 3, 1990

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Pages 1318-1324

May 16, 1990

STOMATOCYTIC

Makoto

OR DISCOIDAL

Nakao,

ERYTHROCYTE SPECTRIN

GHOSTS CONTAINING

Yuko Kojima, Shingo Sato, Kenjiro Wake*

Yukichi

Department of Biochemistry, *Department of Anatomy, and Dental University School of Medicine, Yushima, Tokyo, Japan 113 Received

April

Hara

ONLY

and

Tokyo Medical Bunkyo-ku,

9, 1990

SUMMARY: We extracted Triton-treated erythrocyte ghosts with 2 M KC1 (Triton/KCl/ghosts), and then with 1.2 M KBr at pH 5.5 Triton/KCl/KBr ghosts were very similar (Triton/KCl/KBr ghosts). Triton ghosts and Triton/KCl ghosts in shape to untreated ghosts, under a phase-contrast microscope at various pH values and salt concentrations, despite having lost most of their phospholipids and proteins, except for spectrin. Negatively stained Triton ghosts, Triton/KCl ghosts and Triton/KCl/KBr ghosts appeared similar to each other, but the regularity of the spectrin network structure decreased somewhat in that order. Triton/KCl/KBr ghosts were stabilized by adding both actin and band 4.1, but not by adding either alone. These and previous findings strongly suggest that the spectrin network is visible and the simplest inframembrane structure. 01990 Academic Press, Inc.

flexible and highly The erythrocyte membrane iS quite viscoelastic, and its shape is responsive to changes of pH, salt concentration and various agents including intracellular ATP and calcium ions. Most of these functions are essentially related to those of the inframembrane structure (1). However, the inframembrane structure has more than ten components (see ref. 2), and it has not been clear which component is the most critical one. We have now obtained stomatocytic ghosts containing only spectrin. MATERIALS

AND METHODS

Outdated human packed erythrocytes were provided by the Red Cross Blood Center. Ghosts were obtained and treated with 6% Triton as previously described (1) except for the Triton concentration (Triton ghosts). After washing with 10 mM Tris-Cl buffer, pH 7.4, containing 0.1 mN CaCl,, 1 mM ATP and 20 pg/ml Abbreviations: SDS, sulfonyl fluoride. 0006-291X/90 Copyright All rights

sodium

dodecyl

$1.50

0 1990 by Academic Press, Inc. of reproduction in any form reserved.

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sulfate;

PMSF,

phenylmethyl-

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PMSF, they were extracted with 2 M KCl(Triton/KCl ghosts) and then extracted with 1.2 M KBr in 20 mM Na citrate buffer containing 0.1 mM CaCl,, 1 mM ATP and 20 /.&g/ml PMSF, at pH 5.5 (Triton/KCl/KBr ghosts). The residues after salt treatment were harvested on a discontinuous sucrose density gradient essentially according to Sheet2 (3) with a slight modification. SDS- polyacrylamide gel electrophoresis was performed according to the previous paper (4). 41-54 diameters of ghosts were taken from phase contrast micrographs with an image analyzing system (Ratoc System, Tokyo). Lipid phosphate was assayed according to Fiske and SubbaRow(5). Protein was determined according to Lowry et al. (6). A 2% solution of uranyl acetate from Merck Co. and grids of "carbon substrate 200 A for high resolution" purchased from Ohkenshoji Co. (Tokyo) were used for electron micrography of negatively stained ghosts. The number of Triton/KCl/KBr ghosts remaining after incubation for 15 min at 30°C was counted to observe the stability of the ghosts. The incubation mixture contained 154 mM KCl, 10 mM Tris-Cl buffer, pH 7.4, 15 I.rg of protein 4.1 and 100 pg of spectrin (Triton/KCl/KBr ghosts) in a volume of 500 ccl. Actin and protein 4.1 were obtained according to Spudich and watt (7) and Ralston and Ralston (8), respectively.

RESULTS

AND DISCUSSION

Ghosts treated at low ionic strength with 0.5% Triton X-100 exhibited a cup form under a dark-field microscope as shown in Fig. 2 of our previous paper(l), where we had suggested that the Triton-treated ghosts were crenated. When 6% Triton was used to treat the ghosts instead of 0.5% Triton, small lipid vesicles bound to the inframembrane meshwork disappeared completely. A large concentration of KC1 removed most of band 3, probably including adducin, band 2.1, or ankyrin, band 2.3, band 2.4, band 4.2 and smaller peptide areas including bands 6,7 and 8, but spectrin, protein 4.1, band 4.9 and actin remained. In contrast, protein 4.1, band 4.9, and actin disappeared after but not band 3, band 4.2, etc. treatment with KBr at pH 5.5, Medium pH and a KBr concentration of more than 0.8 M were extraction. Otherwise, the network important factors for the was destroyed or the specificity of the extraction was reduced. treatment with KC1 and then KBr was very effecThus, successive tive for extracting most proteins, with exception of spectrin (Fig. 1). Ankyrin, band 3, adducin, protein 4.1, band 4.2, were clearly observed in the KC1 and KBr protein 4.9, and actin extracts, by the aid of SDS-polyacrylamide gel electrophoresis. Phospholipid was assayed by phosphate determination, assuming content was the average molecular weight to be 800. The lipid 2.2% or less in the Triton/KCl about 16% in the 6% Triton ghosts, ghost and 1% or less in the Triton/KCl/KBr ghosts. As viewed by 1319

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phase-contrast microscopy, both the Triton/KCl and the Triton/KCl/KBr ghosts were very similar in appearance to the original Triton ghosts. They were stomatocytic or discoidal at various pH values, and salt concentrations, as shown in Fig. 1. Diameters of the Triton/KCl ghosts were smaller than those of the original ghosts and Triton ghosts, and those of the Triton/KCl/KBr ghosts were much smaller. These ghosts shrink reversibly at high ionic concentrations and in acidic media (Table 1). The different types of ghosts were negatively stained with uranyl acetate on fenestrated carbon films and observed under an electron microscope. The inframembrane network in the 6% Triton ghosts was periodically arranged, but in the Triton/KCl ghosts, the regularity was less good and in the Triton/KCl/KBr ghosts, it was even worse. Some coiled, tangled strings were observed around the centers from which rather straight spectrin tetramers radiated. One distal end of a spectrin tetramer seemed to be released from another tetramer and then became wound or coiled. It is surprising that a closed mesh or basket consisting of only spectrin is visible under a phase-contrast microscope, even though the thickness of spectrin is very small [5-8 nm (9) or less]. Tsukita et al. demonstrated some years ago (10) that ghosts showed spider-like features after tannic acid fixation: and when the ghosts were previously treated with low concentrations of buffer containing EDTA, the spider-like network disappeared together with actin, spectrin and some other components. The network reappeared on the addition of spectrin alone, as well as when the whole extract was added. We were also able to reconstitute more stable networks. Various amounts of actin were added to the Triton/KCl/KBr ghosts in 140 mM KC1 solution in the presence of a Specific amount of protein 4.1 (molar ratio of spectrin tetramer to protein 4.1, 1:4). The spectrin ghost without protein 4.1 or actin disappeared The addition of completely after incubation at 30°C for 15 min. actin to spectrin at the ratio of I to 5 (spectrin tetramer to actin monomer ratio, close to that in vivo) resulted in 70% Fig. l.Phase contrast microscopy and SDS gel electrophoresis pattern of Triton/KCl/KBr ghosts. Human erythrocyte ghosts were treated with 6% Triton (Triton ghosts, A). After washing, they were first extracted with 2 M XCl(Triton/KCl ghosts, B) and then extracted with 1.2 M KBr patterns of SDS-polyac(Triton/KCl/KBr ghosts, C). Densitometric rylamide gel electrophoresis of A,B,and C are indicated in a,b,and c respectively. Bar=5 pm.

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168, No. 3, 1990 Table

1.

Approximate

AND BIOPHYSICAL

diameter Triton/KCl/KBr Diameter

and

5.5 w 4.0 3.4 2.9

7.4

8.5

6.5 6.2 4.4

10.3* 8.2 5.7

phospholipid ghosts

content Lipid

KC1

DH

Ghosts Triton Triton/KCl Triton/KCl/KBr * at pH 9.5. The average contrast micrographs. 15%.

RESEARCH COMMUNICATIONS

OmM 150m 6.5 6.2 4.4

diameter of 41-54 Each coefficient

content

pmole

P/mg protein 0.236 <0.028
4.1 3.7 3.1 ghosts of

of

was taken variation

from phasewas less than

the ghosts, and at the 1 to 15 ratio more than 90% When various amounts of protein 4.1 were added in the (Fig. 3A). presence of a specific amount of actin (spectrin tetramer to actin monomer ratio, 1 to 5), there was about 70% saturation of the number of the ghosts remained after incubation and a one to seven ratio resulted in more than 90% saturation. The next finding is rather indirect. The spectrin network was destroyed by incubation at various temperatures and in the presence or absence of KCl, and the products were examined by polyacrylamide gel electrophoresis in the absence of SDS. Dimers, tetramers and high order polymers were clearly observed, but bands of oligomers of a slightly higher order than tetramer were absent or very Generally speaking, fragmented erythrocytes or ghosts are weak. but the ghosts containing often observed during their breakdown, only spectrin appeared as slightly small, but complete cells, or were invisible. That half-damaged ghosts were not observed is a retention

of

characteristic

of

100

the

spectrin

ghosts.

A

,

B

,Protein 4.1 wg .

+Actin

25pg

l

3 z! ‘s

50

9

v=0

5s7.I. 10 Added

Fig. 3.Stabilization spectrin. The number tion withsactin tein 4.1 (A) or d. The incubation values are given

100

Actin

( pg/lOOpg

of

Spectrin

)

Triton/KCl/KBr

Added

Protein

ghosts

4.1( pg/lOOpg

Spectrin

containing

)

only

of Triton/KCl/KBr ghosts remaining after incubain the absence or the presence of 15 I.rg of pro25 pg of actin (B) for 15 min at 30°C was countemixture is shown in MATERIALS AND METHODS. as a percent of the original number.

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The above findings strongly suggest that the basic structure of the erythrocyte inframembrane consists only of spectrin, and both actin and protein 4.1 reinforce the structure. Non-erythroid cells have spectrin-like molecules. Whether these molecules play the same role as they do in erythrocytes remains to be established. ACKNOWLED@fENTS This Education,

work was supported by grants Science and Culture, Japan.

from

the

Ministry

of

REFERENCES (l)Jinbu, (2)Bennett, (3)Sheetz, (4)Fairbanks, Biochemistry (S)Fiske, (6)Lowry, J. (1951) (7)Spudich, 4871. (8)Ralston, Acta 775, (9)Cohen, 875-883. (lO)Tsukita, M. (1981)

Y.,

Sato, S., and Nakao, M. (1984) Nature 307. 376-37. V. (1989) Biochim. Biophys. Acta 988, 107-121. M. P. (1979) Biochim. Biophys. Acta =,122-134. G., Steck, T. L., and Wallach, D. H. F. (1971) j&2606-2617. C. H. & SubbaRow, Y. (1925) J. Biol. Chem. 66, 375-400. 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J.Biol. Chem. =,265-275. J. A., and Watt, S. (1971) J. Biol. Chem. 246, 4866C. E., and Ralston, 313-319. C. M., Tyler, J. M., J.

Sa., Cell

Tsukita, Biol. s,

G. P.

(1984)

and Branton,

Sh. Ishikawa, 70- 77.

1324

Biochim. D.

H.,

Biophys.

(1980)

Sato,

S.,

Cell

21,

and Nakao,