Biochemical Society Transactions (1992) 20 1 1 5s Probe and protein orientations in proteoliposomes: electron microscopy and topobiochemistry.

P. NICHOUS, B.TAlTRIE, P.BUTKO & M.TIHOVA

dimenc inlaid enzyme[4]. Fig. 2 shows the quenching behaviour of HC-COV and pyranine-trapped COV. treated with xylene-bispyridinium. Although pyranine is more readily quenched than HC, only the externally bound fraction interacts with DPX. The HC distributes so that 67% is DPX-accessible.

Dept.Biol.Sci.. B m k Univ.,St.Catharines, Ont.L2S3A1,Canada. Cytochrome oxidase-containing vesicles (COV) were prepared by cholate dialysis[l]. Beef heart cytochrome oxidase (uu3) was incorporated into asolectin or a dioleylWdioleylPE lipid mixture, with and without the pH probes pyranine(PY) N o r 4-heptadecyl7-hydroxycoumarin (HC). Five methods were used to determine probe & tau3 orientation. The a 3 incoqmration was analysed by (i) obtaining EM images of oxidase 'knobs' on freeze-fracture convex & concave faces, (ii) reduction with ascorbate, cyt.c and TMPD [2] & (iii) activity measurements before and after lysis of the COV. H W Y orientation was analysed by (iv) passive H+ pulses & (v) fluorescence quenching.

FoIAF

Fig. 2. Pyranine and HCCOV auenchlnrr bv DPX.

5-019 pyranine

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I

2.5

cutldese distribution and in different DFAF -rf of COV, RC' Orientation# TN ea3:PL ratio l w e c l Lper sec) 3.4 78 73 420 0.39

Table 1. Qtochrome Sample Total DEAE 1

7

PO

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-0

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DEAE-fractionation of COV showed oxidase distributed differently in different populations (Table I). The smallest vesicles contain either no enzyme or only inward-facing enzyme. Larger vesicles contain both inward- and outward-facing enzyme. The average distribution corresponds to =75% outward- & 25% inward-facing by all 3 techniques (Table I). As the freeze-fracture plane passes centrally through the membrane the visible 'knobs' include complexes in both orientations; the average protein density by occupied area is the same whether convex or concave fracture planes are analysed. However, average heights of protruding species differ in convex and concave faces. More 67nm knobs are seen on convex faces, and more 3.5-4.0nm knobs on concave faces. We conclude that the former are oriented with their 'C' faces protruding from the fracture plane and the latter with their 'M faces protruding. With this assumption the ratios correspond reasonably well to the 75%:25% ratio obtained chemically or enzymically. Cytochmme oxidase was then depleted of subunit III as well as some smaller subunits, essentially according to [3]. Fig. 1. EM areas of control & sulll-depleted enzyme 301 n 1

6

20

e al a

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E

1

I

.

I

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0.5 1.o 1.5 2.0 l/[DPXJ(a) & POI[DPX](b) Asoiectin COV prepared with 5mM trapped pyranine probes, or with 3mol% heptadecylhydroxycoumarin

0.0

0.003

2.7 71 500 m Resp. control(response to valinomycln+nigericin) # K enzyme facing outwards, by lauryi maitoslde rctlvetlon(act) or reductant accessibiiity(spec). a"ry ed

WAF 2

0.0

10

0

Particle areas from Pt-shadowed freeze-fractured DOPE-DOPC COV(1ipid:protein ratio=lO), DEAE n#2 : c-ces. - D

Fig. 1 shows the distribution of 'knobs' by area; depleted enzyme occupies -50% the surface area of undepleted enzyme. The average decrease in monomer mass is however only about 17% for an expected area decrease of no more than 11%. The observed effect is thus likely to represent monomerization of an initially

measurements and the DPX quenching data give 33% HC in and 67% HC out. The similarity of probe (Table II) and protein (Table I) incorporation ratios indicate essentially random incorporation of uu3 & HC according to internally and externally accessible COV area. It is not necessary to assume a preference by the oxidase for membranes of specific curvature or any kind of ordered enzyme incorporation into preformed membranes[S].There are, however, consequences for active measurements of pH gradients generated by enzyme turnover.

sed or s o w d COV, Yesicles

% Quench'

pyranine