Binding of markers of either side of plasma membranes to the cytoplasmic side of lysosomes

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Experimental Cell Research 110 (1977) 103-l 10

BINDING MEMBRANES

OF MARKERS

OF EITHER

TO THE CYTOPLASMIC Y. RIKIHISA

Faculty of Pharmaceutical

SIDE OF PLASMA SIDE OF LYSOSOMES

and D. MIZUNO

Sciences, University

of Tokyo, Hongo,

Tokyo, Japan 113

SUMMARY The binding of markers of each side of plasma membranes of guinea pig polymorphonuclear leukocytes (gp-PMNL) to the cytoplasmic side of membranes of isolated lysosomes of gp-PMNL was studied using an improved indirect immunoferritin technique. Anti-cell surface IgG (anti-C-S IgG) which bound to the outer surface of intact cells and was eluted from a complex of cells and antibody was previously shown not to bind to the cytoplasmic side of open sheets of plasma membrane of gp-PMNL [2]. Electron microscopy using the indirect immunoferritin technique showed that this antibody bound to the cytoplasmic side of lysosomes. Non-immunized rabbit IgG did not cause binding of indirect ferritin conjugate to lysosomes. On the other hand, influenza virus, which also bound exclusively to the outer side of open sheets of plasma membrane [2], did not bind to the cytoplasmic side of lysosomes under conditions where it bound to contaminating membrane vesicles. Anti-100000 g sup IgG which was prepared against the 100000 g sup of a gp-PMNL homogenate in 0.6 M KC1 and was preabsorbed with intact cells, i.e., which did not contain antibodies against the outer side of intact cells, bound specifically to the cytoplasmic side of open sheets of plasma membrane [2]. This antibody also bound to the cytoplasmic side of lysosomes. Moreover lysosome-specific antigenicity, i.e., antigenicity that was not common to plasma membranes, was found to be present on the cytoplasmic side of lysosomes. These results show similarities in the membrane components of plasma and lysosomal membranes, but also demonstrate great topological differences between the components.

As a part of a series of investigations on formation of phagolysosomal membranes from plasma and lysosomal membranes, we are now locating the markers of each side of the plasma membrane on lysosomal membranes by binding studies using indirect immunoferritin conjugate. Immunoferritin binds specifically because it has been possible to remove contaminating material showing non-specific binding from it [l]. Despite the importance of lysosomal function, there are only a few studies on lysosomal membranes; there are a few reports of comparative studies on the sugar residues, lipids and enzyme activities of plas-

ma and lysosomal membranes [3-6], but the locations of these components on the membranes of lysosomes are unknown. For studies on membrane components using colloidal iron, Henning et al. suggested the presence of sialic acid on the inner side of lysosomes and it did not on the outer side of lysosomes [7]. Feigenson et al. [8] reported the presence of ricin-binding sites on the cytoplasmic surface of lysosomes. However, neither group reported any evidence on plasma membranes by the same procedure. In this study we examined lysosomal membranes using three already characterized markers of the two sides of the Exp

Cell

Res

II0

(1977)

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Rikihisa and Mizuno

Figs l-5. Bar, 0.5 pm, X57000. Fig. 1. The lysosome fraction was incubated with 70 pg of anti-C-S IgG (rabbit). Then it was washed and

incubated with 300 pg of ferritin labeled anti-rabbit IgG (goat). Incubations were carried out for 20 min at 25°C in 0.34 M sucrose.

plasma membrane. For quantitative purposes the numbers of fenitin particles per unit length of membrane were counted in randomly selected photographs of the membranes .

0.6 M KC1 soluble gp-PMNL homogenate and preabsorbed with intact cells. Antibodies were located using improved indirect immunoferritin, which had been freed from materials causing non-specific binding by DEAE-cellulose column chromatography and preabsorption with the membrane fraction [l].

MATERIALS

AND METHODS

gp-PMNL was obtained from peritoneal exudates induced by injection of casein, as described previously [2]. The preparation and characterization of the markers which bind specifically to one or the other side of the plasma membranes were described previously. Anti-gp-PMNL antiserum was prepared by injection of intact gp-PMNL into rabbits; it had a higher titer aaainst intact an-PMNL than that of antiserum obtained- by the conventional method. This antiserum was treated with intact gp-PMNL and bound antibody was detached from the cells and purified (anti-C-S IgG). Inthtenza virus A/NWS (HONl) was grown in eggs and purified by centrifugation on a linear sucrose gradient. Anti-100000 g sup IgG was prepared against Exp Cell Res I IO (1977)

Isolation

of the lysosome fraction

Cells were disrupted by pipetting them back and forth in 500 U of heparin/ml of 0.25 M sucrose and the disrupted cells were fractionated by the procedure of Bell et al. [9]. The lysosome fraction was divided into five equal parts (about 2 mg of protein each) which were treated at the same time with anti-C-S IgG, anti100000 g sup IgG, anti-lysosome IgG, virus and nonimmunized IgG as a control, respectively.

Preparation

of anti-lysosome

ZgG

Japanese albino rabbits were sensitized by 13 subcutaneous or intravenous injections of a total of 8.2 mg of protein of lysosome fraction of gp-PMNL over a

Cytoplasmic side of lysosomes

105

Reaction of the lysosome fraction with anti-C-S ZgG or other ZgG and then with immunoferritin

Fig. 2. The procedure was the same as for fig. 1 except that 120 pg of non-immunized IgG (rabbit) was used instead of anti-C-S IgG.

Lysosome fractions were prefixed with 2% paraformaldehyde at 4°C for 20 min. This treatment did not decrease the amount of bound antibody. Then the prefixed fractions were incubated with rabbit antibodies (anti-C-S IgG, anti-100000 g sup IgG, anti-lysosome IgG or non-immunized IgG) at 25°C for 20 min. Unbound antibodies were removed bv washing three times with 0.34 M sucrose, 1 mM Tris-HCI buffer, pH 7.0 with centrifugation at 25000 g for 10 min at 4‘C. Then an equal amount of improved indirect antirabbit IgG goat IgG-ferritin conjugate [l] was added to each antibody-treated fraction and the mixtures were incubated at 25°C for 20 min. Unbound ferritin conjugate, was removed by washing and the lysosome fractions were prepared for electron microscopy. Influenza virus was incubated with lysosome fractions at 4°C for 20 min and unbound virus was removed by centrifugation.

period of 5 weeks, following the method for preparation of anti-gp-PMNL serum [2]. The IgG fraction was obtained from this anti-lysosome antiserum by precipitation with Na$O, and DEAE-cellulose chromatography.

Electron microscopy

Fig. 3. The lysosome fraction was incubated with influenza virus for 1 h at 4°C in 0.34 M sucrose and then

centrifuged at 25000 g for 10 min to remove unbound virus.

After the above treatments, the lysosome fractions were fixed in 2% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4 at room temperature for 12h. Then they

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Rikihisa and Mizuno

Fig. 4. A lysosome preparation was incubated with 600 pg of anti-108 000 g sup IgG and then washed and incubated with 300 pg of ferritin labeled anti-rabbit

IgG (goat). Incubations were carried out for 20 min at 25°C in 0.34 M sucrose.

were washed and fixed in 1% 0~0, in 0.1 M cacodylate buffer at room temperature for 1 h. They were stained as blocks with 4 % many1 acetate, dehydrated in a graded series of ethanols and embedded in EponAraldite mixture (TAAB Laboratories. Readine. UK). Sections were stained with 4 % many1 acetate &d lead acetate and examined in a JEM-1OOB electron microscope operated at 80 kV.

cifically to the outer surface of open sheets of plasma membrane [2]. In this work, we found by the indirect immunoferritin technique that this antibody bound to the cytoplasmic side of lysosomes (fig. 1). Ferritin particles bound exclusively to one side of contaminated membrane vesicles too. When non-immunized IgG was used instead of anti-C-S IgG, little non-specific binding of indirect immunofenitin to the lysosome preparation was observed (fig. 2). This was due to the presence of antigenicities common to both the outer cell surface and the cytoplasmic side of lysosomes.

RESULTS Binding of anti-C-S IgG

In previous studies using immunoferritin we found that anti-C-S IgG which had bound to the outer surface of intact cells and then been detached from the cells, bound speExp Cdl RPS I IO ( 1977)

Cytoplasmic

side of lysosomes

107

Fig. 5. The procedure was as for fg 4 except that about 100 wg of anti-lysosome IaG was added instead of 600 pg of anti-100000 g sup IiG.

Binding of influenza

virus

Previously we showed that influenza virus bound specifically to the outer surface of open sheets of isolated plasma membrane [2]. Fig. 3 shows that under conditions in which the virus bound to contaminating membranes, it did not bind to lysosomes. Thus a virus-binding site was not present on the cytoplasmic side of lysosomes. However, anti-C-S IgG, another marker of the outer side of plasma membranes did bind to the cytoplasmic side of lysosomes. Thus, the components of the outer cell surface and the cytoplasmic side of lysosomes were not all identical. Binding

of anti-100000 g sup ZgG

Anti- 100000 g sup IgG was prepared against

the 100000 g sup of a homogenate of gpPMNL in 0.6 M KC1 and preabsorbed with intact cells. This antibody specifically to the cytoplasmic side of open sheets of isolated plasma membrane [2]. The anti100000 g sup IgG was found to bind to the cytoplasmic side of lysosomes (fig. 4). Thus, the cytoplasmic sides of plasma and lysosomal membranes have some common antigenicity. This antigenicity was not found on the cytoplasmic side of phagolysosomes, as shown in another paper [lo]. Binding of anti-lysosome

Anti-lysosome IgG the crude lysosome sorbed with intact membrane fraction.

ZgG

was prepared against fraction and then abcells and the plasma This preparation was Exp Cell Res 110 (1977)

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Rikihisa and Mizuno

Table 1. Average number of granules of ferritin-labeled anti-rabbit IgG goat IgGbound to the cytoplasmic side of lysosomes in electron micrographs Rabbit IgG

Total membrane length 6-4

Total no. of bound ferritin particles

No. of ferritin particles bound per pm of membrane

Anti-C-S IgG Anti- 100000 g sup IgG Anti-lysosome IgG Non-immunized IgG

23.5 15.1 12.0 11.0

739 803 178 68

31.4 53.2 14.8 6.2

also found to bind specifically to the cyto- performed in parallel using the same lysoplasmic side of lysosomes (fig. 5) and not to some preparation and the same anti-rabbit contaminating membrane vesicles. IgG goat IgG-labeled with ferritin. With Quantitative measurements of the bind- non-immunized IgG, few ferritin particles ing of indirect ferritin conjugate after reac- were bound (fig. 2, table 1). In contrast, tion with anti-C-S IgG, non-immunized with anti-C-S IgG, anti- 100000 g sup IgG or IgG, anti-100000 g sup IgG or anti-lyso- anti-lyososome IgG, significant numbers of some IgG were achieved by counting the ferritin particles were observed on the cytogranules of ferritin within a radius of 40 nm plasmic side of lysosomes (figs 1,3,4 and 5, of membrane in each about 20 sheets of table 1). These findings show that anti-C-S photographs, respectively. The results are IgG, anti-100000 g sup IgG and anti-lysosummarized in table 1. Reactions with the some IgG bind specifically to the cytofour rabbit antibodies were performed si- plasmic side of lysosomes. Thus anti-C-S multaneously. As expected, the four prep- IgG, which bound to the outer surface of arations which differ only with respect to intact cells and was then detached from the direct rabbit IgG, showed specific binding cells, bound to the cytoplasmic side of lysoof anti-C-S, anti-100000 g sup IgG and anti- somes (fig. 1). The ricin-binding site has lysosome IgG. When the same amount of also been shown to be present on the cytoexcess indirect anti-rabbit IgG goat IgG la- plasmic side of lysosomes [8], but its locabeled with ferritin was added to the four tion on plasma membranes is unknown. Our samples, different amounts of bindings of results show similarities and also great anti-rabbit IgG goat IgG-labeled with ferri- topological differences between the compotin were observed on electron micrographs nents of lysosome and plasma membranes. depending on the amount of binding of each We expected to find that the outer side of rabbit IgG. lysosome membranes was the same as the outer side of plasma membranes, because DISCUSSION in previous work we found that anti-C-S In this paper isolated lysosome membrane IgG bound to the cytoplasmic side of phagowere compared with isolated plasma mem- lysosomes and that the amount of binding of branes using specific markers of the two anti-C-S IgG increased in parallel with sides of plasma membranes with an im- fusion with lysosomes [IO]. However, alproved indirect immunoferritin technique. though the outer surface of plasma memExperiments on different rabbit IgG were branes and the cytoplasmic side of lysoExp Cell Res 110 (1977)

Cytoplasmic side of lysosornes somes share common antigenicities, their components are not identical, because lysosome membranes have their own specific antigenicity (fig. 5), they differ from the outer surface of plasma membranes in binding anti- 100000 g sup IgG and in not binding influenza virus. The topology of antigenicity against anti-100000 g sup IgG and sialic acid residues seems to be similar in plasma membranes and lysosomal membranes. Henning et al. also showed that the outer side of liver lysosomes did not stain with colloidal iron, which was believed to stain sialic acid residues [7]: they did not report data on plasma membrane. Furthermore we found using anti-gp-PMNL myosin IgG and improved immunoferritin [12] that gp-PMNL myosin is located on the cytoplasmic side of plasma membranes, but not on the cytoplasmic side of lysosomes (data not shown). These results show that some components of the outer surface of plasma membranes are present on the cytoplasmic side of lysosomes. This suggests that formation of these membranes is not entirely due to the process of membrane flow [ll]. Fur-

109

ther studies on the formation of phagolysosomal membranes are reported in the preceding paper [lo]. REFERENCES 1. Rikihisa, Y, Ohkuma, S & Mizuno, D, Cell structure and function 1 (1976) 251. 2. - Exp cell res 100 (1976) 23. 3. Kaulen, H D, Henning, R & Stoffel, W, HoppeSeyler’s Z physiol Chem 351 (1970) 1555. 4. Henning, R, Kaulen, H D & Stoffel, W, HoppeSeyler’s Z physiol Chem 351 (1970) 1191. 5. Henning, R & Heidrich, H, Biochim biophys acta 345 (1974) 326. 6. Thines-Sempoux, D, Lysosomes in biology and pathology (ed J T Dingle) vol. 3, p. 278. NorthHolland, Amsterdam (1973). 7. Henning, R, Plattner, H & Stoffel, W, Biochim biophys acta 330 (1973) 61. 8. Feigenson, M E, Schnebli, H P & Baggiolini, M, J cell bio166 (1975) 183. 9. Bell, D A, Theiem, P A, Vaughan, J H & Leddy, J P, J clin invest 55 (1975) 256. 10. Rikihisa, Y & Mizuno, D, Exp cell res 110 (1977) 93. 11. Siekevitz, P, Palade, G E, Dahner, G, Ohad, I & Omura, T, Organizational biosynthesis (ed H J Vogel, J 0 Lampen & V Dryson) p. 331. Academic press, New York (1967). 12. Rikihisa, Y & Mizuno, D, Exp cell res 110 (1977) 87. Received March 16, 1977 Revised version received June 22, 1977 Accepted July 8, 1977

Exp CellRes 110 (1977)