Transglutaminase modification of rhodopsin in retinal rod outer segment disk membranes

ARCHIVES

Vol.

OF BIOCHEMISTRY

249, No. 2, September,

AND

BIOPHYSICS

pp. 506514,1986

Transglutaminase Modification of Rhodopsin in Retinal Rod Outer Segment Disk Membranes’ J. HUGH

MCDOWELL,? AND

ANDREA UBEL,* RANDAL PAUL A. HARGRAVEP’

A. BROWN,*

*Department of Medical Biochemistl-y, Southern Illinnis University School of Medicine, Carbondale, Illinois 62901, and tDepartments of Ophthalmology and Biochemistry, University of Florida School of Medicine, Gainesville, Florida 32610 Received

February

28,1986,

and in revised

form

May

12,1986

Rhodopsin in rod outer segment disk membranes was enzymatically modified by erythrocyte transglutaminase, which linked small primary amines to glutamine residues. In order to avoid formation of protein crosslinks, rhodopsin was first reductively methylated to modify its lysines. From 1.9 to 2.5 mol of putrescine, ethanolamine, or dinitrophenylcadaverine were incorporated into rhodopsin by transglutaminase during 16 h reaction time. A maximum of 3.5 mol of [14C]putrescine was incorporated per mole of rhodopsin during 48 h. Essentially all of the rhodopsin sequence containing the putrescine could be removed by limited proteolysis of the membranes by thermolysin. Glutamine residues in positions 236, 237, 238, and 344 were modified to approximately equal extents, as determined by isolation of the cyanogen bromide peptides of modified rhodopsin followed by further subdigestion of the peptides. The modified glutamine residues are located in the helix V-VI (or Fl-F2) connecting loop and in the carboxylterminal region of rhodopsin. o 1986 Academic press, Inc.

Rhodopsin is the photoreceptor protein of rod cells in the retina. It is a well-studied integral membrane protein whose sequence is now known for cattle (24, 13, 21), sheep (2), humans (22), and Drosophila (23, 29). On the basis of extensive topographic studies [reviewed by Hargrave (9)] and predicted secondary structure, similar models for rhodopsin’s topography in the disk membrane have been proposed (24,5, 25). Experiments which have been important for the identification of exposed regions of the polypeptide chain have employed chemical modification of rhodopsin or modifications with proteases or with transglutaminase.

Rhodopsin in rod outer segment membranes can be modified in a protease-sensitive region by incorporation of primary amines into glutamine residues by means of transglutaminase (26). Evidence has been presented suggesting that the modification occurs to an extent of 1 mol of primary amine per mole of rhodopsin under conditions which result in some crosslinking of rhodopsin to itself, and that the incorporation occurs in the Fl-F2 (or helix V-VI) connecting loop (26). This loop region is located at the cytoplasmic or extradiskal surface of rhodopsin and is highly accessible to the aqueous environment. The cytoplasmic surface of rhodopsin is important in rhodopsin’s function. G-protein (transducin) binds to rhodopsin’s surface following exposure to light, an early step in the visual transduction process (27). Other proteins, rhodopsin kinase and a M, 48,000 protein, also interact with rhodopsin’s surface after rhodopsin has been ex-

i Supported in part by National Institutes of Health Grants EY 06225 and EY 06226, an unrestricted departmental grant from Research to Prevent Blindness, Inc., and a Jules and Doris Stein Research to Prevent Blindness Professorship Award (to P.A.H.). a To whom correspondence should be addressed. 0003-9861/86 Copyright All rights

$3.00

0 1986 by Academic Press, Inc. of reproduction in any form reserved.

506

TRANSGLUTAMINASE-MODIFIED

posed to light (15). Because of the functional significance of this region of rhodopsin, it is of importance to further study and securely describe its topography. Use of transglutaminase may also allow US to prepare defined derivatives of rhodopsin which will be useful in probing the function of various surface regions. In the present study we have determined the specific sites on rhodopsin’s surface which become modified by the addition of small amines due to the action of transglutaminase. MATERIALS

AND

METHODS

Materials. Outdated human blood was obtained from the American Red Cross Blood Service. Acrylamide bisacrylamide, TEMED: ammonium persulfate, SDS, and protein molecular weight standards for gel electrophoresis were purchased from Bio-Rad Laboratories. N-Bromosuccinimide was purchased from Sigma Chemical Corporation and was recrystallized from water before use. SAV8 protease was purchased from Miles Laboratories. Sequenal grade PITC, TEA, and TFA were from Pierce Chemical Company. Bovine serum albumin (radioimmunoassay grade) was purchased from the Sigma Chemical Corporation. Transglutaminase preparation. Transglutaminase was prepared from red blood cells by the method of Brenner and Wold (1). The enzyme preparation obtained following gel filtration on Bio-Gel A-O.5 m was used without further purification. Protein concentration in the enzyme preparation was determined by using the Bio-Rad protein assay system with bovine serum albumin as standard. Preparation of Me,ROS Rod outer segments were prepared from frozen retinas (American Stores Packing, Inc.), as described earlier (19). All procedures were carried out under dim red light unless noted otherwise.

’ Abbreviations used: Hepes, 4-(2-hydroxyethyl)-lpiperazineethanesulfonic acid; PITC, phenylisothiocyanate; DNP-, 2,4-dinitrophenyl-; TrTAB, tridecyltrimethylammonium bromide; ROS, rod outer segments; Me2ROS, reductively methylated rod outer segments; SDS, sodium dodecyl sulfate; TEMED, N,N,N’,N’-tetramethylethylenediamine; NBS, Nbromosuccinimide; TEA, triethylamine; TFA, trifluoroacetic acid; TCA, trichloroacetic acid; Con A, concanavalin A; Con A buffer, 50 M Tris-Ac, 1 mM MgCIZ, 1 mM MnC12, and 1 mM CaCl,, pH 6.9; PAGE, polyacrylamide gel electrophoresis; SAV8 protease, Staphylococcus aureus, strain V8, protease; FDNB, lfluoro-2.4-dinitrobenzene; DTE, dithioerythritol.

RHODOPSIN

507

The soluble proteins were extracted by homogenizing the ROS on ice in 2 mM sodium phosphate, 1 mM MgC&., 1 mM DTE, and 0.1 mM EDTA (pH 7.0) at a rhodopsin concentration of about 10 mg/ml. The membrane fragments were collected by centrifugation at 31,300g for 30 min. Reductive methylation of the membranes followed the procedure of Jentoft and Dearborn (14). The ROS membranes were suspended at 4°C in 0.1 M Hepes buffer (pH 7.5) at a concentration of about 1 mg/ml rhodopsin. Excess formaldehyde, approximately 35,000 times the rhodopsin concentration, was added as a 37% solution and the pH was adjusted to 7.5 with 0.2 M NaOH. NaCNBH$, 1 M, was added to yield a final concentration of 20 mM. The reaction mixture was stirred in the dark at 4°C for 24 h. The Me,ROS were then collected by centrifugation. Transglutaminase modijcation of Me&OS. The Me,ROS were washed three times with 0.1 M Tris-Ac, 20 mM CaC&, and 5 InM DTE (pH 7.4). Labeling buffer was prepared by making the above buffer 10 mM in amine substrate and adding radioactive amine substrate. Samples of the buffer were taken for specific activity determination before the MezROS were suspended to about 10 mg/ml rhodopsin. Transglutaminase concentrate was added to yield a 1:lO weight ratio of transglutaminase to rhodopsin. The reaction mixture was incubated at 37°C in the dark with stirring overnight, typically about 16 h. The labeled membranes were collected by centrifugation. In order to determine the kinetics of putrescine incorporation, triplicate 507.11 samples were removed at various times after addition of the transglutaminase and added to 1 ml of ice-cold 10% TCA in 1.5-ml snaptop centrifuge tubes. Washing and counting of the samples was performed as described for phosphorylated rhodopsin (28). Afinity puri&atinn using Con A-Sepharose. Transglutaminase-modified rhodopsin or its Fl-F2 complex resulting from limited proteolysis was purified by affinity chromatography on Con A-Sepharose as described earlier (18) except that TrTAB was used instead of octyl glucoside. The ROS membranes were dissolved in 0.2 M TrTAB made in Con A buffer, and all remaining chromatography buffers contained 0.1 M TrTAB in Con A buffer. In the purification of the carboxyl-terminal cyanogen bromide peptide (CB3, residues 321-348), the glycopeptide present (CBl, residues 2-39) was removed by chromatography on Con A-Sepharose. The procedure of Mas et aL (18) was followed except the buffers contained no detergent. Under these conditions peptide CB3 passes through the column without binding and is then desalted by chromatography on Bio-Gel P-2 as described below. Protein chemistry. Affinity-purified modified rhodopsin was dialyzed to remove detergent, aminoethylated, and cleaved with CNBr (12). Size fractionation of the CNBr peptides was obtained by chromatography

508

MC

DOWELL

on Sephadex G-50 in 20% formic acid (12). Cleavage at tyrosine and tryptophan residues was achieved by using NBS as described by Bustin and Cole (3), except that the cleavage was done in 80% acetic acid. Size fractionation of small peptides and desalting of large peptides were performed by chromatography on a column containing Bio-Gel P-2 (1 X 125 cm) in 0.1 M NH4HC03, pH 7.8. Fractions (1 ml) were collected and aliquots were tested for radioactivity and/or amino acid content (after hydrolysis) by high-voltage paper electrophoresis (4). We performed limited proteolysis of the ROS membranes with thermolysin in order to generate the membrane-bound Fl-F2 complex (12). The digestion mixture was incubated in the dark for 10 h at 3’7°C with 7% by weight of thermolysin, and the membranes were recovered by centrifugation. Subdigestion of peptides with SAVS protease was performed in 0.1 M NHIHCOa, pH 7.8, by using 5% by weight of SAV8 protease. The digestion mixture was incubated at 37°C for at least 10 h. Amino acid analyses were performed on a Beckman 119CL amino acid analyzer following the procedure of Fauconnet and Rochemont (7). Automated sequence analysis was performed on a Sequemat Model 12 sequenator equipped with a P-6 autoconverter. Procedures outlined in the manual were followed except that benzene was used as the solvent instead of dichloroethane, 10% triethylamine in a 3:2 MeOH:water mixture was used as the buffer, and the PITC concentration was increased to 10% in acetonitrite. Peptides were linked to aminopolystyrene by using the water soluble carbodiimide technique with unblocked peptides described in the Sequemat manual. Preparation of [SH]DNP-codavtine. Cadaverine (8 mg of free base) was dissolved in 1 ml benzene. Aliquots (250 ~1) of [aH]l-FDNB (Amersham, 14 mCi/ mmol, 1 mCi/ml in benzene) were added with stirring every 10 min until 5 ml had been added. This resulted in a molar ratio of cadaverine to FDNB of 219. In some preparations, nonradioactive FDNB was added along with the [aH]FDNB, however, the molar ratio of cadaverine to FDNB was kept higher than 7 in order to avoid formation of di-DNP-cadaverine. The reaction mixture was dried under a stream of Nz, dissolved in 250 ~1 of 6.7% formic acid, and subjected to high-voltage paper electrophoresis (Gilson Model DW electrophorator) for 1 h at 6000 V, 4O”C, with 6.7% formic acid (pH 1.6) as buffer. Under these conditions, the [3H]DNP-cadaverine migrates about 26 cm. After drying, the yellow [3H]DNP-cadaverine was eluted from the paper with deionized water. The specific activity was determined from the radioactivity and Es2 = 17,400. The eluted rH]DNP-cadaverine was concentrated to a convenient volume by evaporation under a stream of Nz and stored frozen. An average yield for the synthesis and purification, based on rH]FDNB, was about 80%.

ET

AL. RESULTS

In order to prepare rhodopsin for transglutaminase modification, we modified ROS membranes by reductive methylation using formaldehyde. This reaction was complete, resulting in incorporation of 19.9 mol [3H]formaldehyde per mole of rhodopsin; i.e., 2 mol formaldehyde were incorporated for each of rhodopsin’s 10 free lysine tamino groups. Such a modification of rhodopsin was necessary in order to prevent unwanted side reactions of ROS amino groups catalyzed by transglutaminase. Rhodopsin retained its spectral integrity (as measured by absorbance at 498 nm) following this procedure, and the reductively methylated rhodopsin chromatographed normally on Con A-Sepharose. Nonradioactive formaldehyde was used under identical conditions to modify rhodopsin in ROS membranes which were to be used for modification with transglutaminase. Rhodopsin in reductively-methylated ROS membranes is a good substrate for transglutaminase-catalyzed modification with primary amines. In separate experiments we incorporated three different radiolabeled primary amines into rhodopsin over a 16-h period. [‘4C]Ethanolamine was incorporated to an extent of 1.9 mol per mole of rhodopsin while 2.5 mol of either [3H]DNP-cadaverine or [14C]putrescine were incorporated per mole of rhodopsin. One of these amines, [14C]putrescine, was employed for study of the time course of incorporation. Putrescine is rapidly incorporated into reductively methylated rhodopsin (Fig. 1). Enzymatic modification appears to level off under our conditions at -2.5 mol incorporated after 6-8 h. However, if the reaction is allowed to proceed there is a slow and continuous incorporation of amine lasting for at least 48 h, which results in as many as 3.5 mol of putrescine incorporated per mole of rhodopsin. These results suggest that more than three glutamine residues in rhodopsin are available for reaction with transglutaminase. We have employed limited proteolysis of [‘4C]putrescine-labeled MezROS membranes as one way to assessthe location of

TRANSGLUTAMINASE-MODIFIED

I..,.

0

2

4

6

8

48

TIME (h)

FIG. 1. Time course of transglutaminase-catalyzed incorporation of [‘%]putrescine into MeaROS. Samples at various times of incubation in the dark at 37°C were precipitated, washed, and counted as described under Materials and Methods. Bars are SEM of six individual samples derived from triplicates of two experiments. The point at 48 h was calculated from the after purification of the laOJL nm and radioactivity beled rhodopsin on Con A-Sepharose.

putrescine within the rhodopsin molecule. Digestion of the modified rhodopsin with thermolysin results in complete conversion of rhodopsin to its fragments Fl and F2 (Fig. 2A). It is also noted that both rhodopsin and its fragments are single bands on the SDS gels, and thus crosslinking by transglutaminase has been avoided. In order to determine the degree of retention of [‘4C]putrescine with the Fl-F2 complex, both the proteolyzed and unproteolyzed membranes were solubilized and subjected to chromatography on Con A-Sepharose. In this particular preparation, 3.1 mol of putrescine were incorporated per mole of rhodopsin as determined by the radioactivity eluting with the 0D498 .,-absorbing peak (Fig. 2B). Following proteolysis by thermolysin, only 6% of the [14C]putrescine was retained in the Fl-F2 complex (Fig. 2C). Digestion by thermolysin removes rhodopsin’s 21 carboxyl-terminal amino acids and a small portion of polypeptide chain connecting helices V and VI [(26), reviewed in (12)]. This result suggests that glutamines remaining in the thermolytic

RHODOPSIN

509

Fl-F2 complex are not major sites of modification. In order to determine the precise sites of incorporation of putrescine into rhodopsin, we cleaved another sample of modified purified rhodopsin with CNBr. The peptide mixture was partially separated by column chromatography (Fig. 3A). Seventy-two percent of the radioactivity eluted in the void volume; this region of the column eluate contains aggregated peptide CBVlb (residues 208-253) which includes the helix V-VI connecting region (13). However, 17% of the radioactivity eluted in the position of the carboxyl-terminal peptide, CB-3 [residues 321-348 (12)]. Most of the remaining radioactivity eluted in the position of the CNBr partial cleavage product, [CB6-CB3 residues 310-348 (ll)]. Peptide CB3 was purified from pool c (Figure 3A), yielding a radioactive peptide with the expected composition for residues 321-348 (Table I). There was 0.48 mol [14C]putrescine per mole of peptide, which represents 20% of the specific radioactivity of the rhodopsin in this preparation. Peptide CB3 was digested with SAV8 protease and the peptide mixture fractionated by gel filtration. Amino acid analysis of the radioactive peak (Table I) was consistent with the composition of the expected SAX3 peptide, residues 342-348. There was 0.50 mol [14C]putrescine present per mole of peptide (21% of the specific activity of modified rhodopsin). Since the only carboxyl-terminal glutamine residue is Glna4, we conclude that transglutaminase incorporated putrescine into GlnX4 in one-half of the rhodopsin molecules in this preparation. We suspected that the remaining modified glutamines would be located in the peptide loop connecting helices V and VI and designed our subsequent experiments accordingly. In order to isolate the peptide containing the remaining incorporated putrescine, the CNBr peptides in radioactive pool a (Figure 3A) were cleaved with NBS and subjected to size fractionation (Fig. 3B). Essentially all of the radioactivity eluted as a single peak in a position expected for NBS peptide 224-253. This peptide is the main component of the pool as

510

MC

DOWELL

ET

AL.

A

R-

-

36k

FI -

-

26k

F2

-13k

-

l.O-

10000

I:

B

.WOOs B

OS-

? b LD

Oh-

‘6000 -

I .4000

i

0.4-t

I 5

10

15

20

25

FRACTION 0.5

30

35

40

43

NUMBER

1c

o.*? b

4ooa i

0.3.

!? II

3ooo

I 14

2ooo

I

(D t 8

0.2.

0.1-

5

10

15

20

FRACTION

25

30

35

40

45

NUMBER

FIG. 2. Effect of limited proteolysis on transglutaminase-[“Clputrescine-labeled ROS membranes. (A) SDS-PAGE of labeled MezROS before (1) and after (2) limited proteolysis using thermolysin. SDS-PAGE was performed according to Fairbanks et al. (6). (B, C) Con A-Sepharose affinity chromatography of labeled MezROS before (B) and after (C) limited proteolysis using thermolysin. R, rhodopsin; Fl and F2 are membrane-bound proteolytic fragments of rhodopsin.

TRANSGLUTAMINASE-MODIFIED

CB3

VOID VOLUME

INCLUDED VOLUME t

7’1

t

3000 7

511

RHODOPSIN

A

2400-

bOO-

50

100

150

VOID VOLUME bOO-

200 250 300 TUBE NUMBER M.W.

t

350

400

450

I 500

350

400

450

I 500

3100 t

B 500

400

Fi;JiL

50

loo

150

200 250 300 TUBE NUMBER

FIG. 3. (A) Size fractionation of CNBr peptides of MezROS labeled by transglutaminase with [Wlputrescine. The transglutaminase-[‘%]putrescine-labeled MeaROS were dissolved in detergent; rhodopsin was purified on Con A-Sepharose, reduced and aminoethylated, cleaved at methionine residues with CNBr, and then subjected to size fractionation on Sephadex G-50 in 20% formic acid (see Materials and Methods for details). Fractions were pooled as shown, on the basis of radioactivity. For treatment of fraction a see (B). For fraction c see Table I. (B) Size fractionation of NBS cleavage products of the CNBr peptides in pool a. The [‘*C]putrescine-containing fractions were pooled as indicated and subjected to amino acid analysis (Table I).

indicated by amino acid analysis (Table I) and contains 1.95 mol [14C]putrescine per mole of peptide. There are four glutamines in this peptide, at positions 236, 237, 238, and 244. However, Gln 244is one of the glutamines in the thermolytic Fl-F2 complex. This complex was nearly devoid of [14C]putrescine and thus GlnM is not a candidate for modification. In order to determine the distribution of radioactivity

among the remaining glutamines, solid phase sequencing was attempted. The peptide did not undergo the Edman degradation, presumably having become blocked as a result of the chemical cleavage steps. Attempts designed to deformylate the peptide did not restore its free amino terminus. Further digestion of the NBS-peptide was performed with SAVB protease in order to yield peptide 233-239, Ala-Ala-Ala-Gln-

512

MC TABLE

AMINO

ACID COMPOSITION

Carboxylterminal” CNBr peptide (CB3; residues 321-348) Asx =3 (3)d Thr 5.72 (6) Ser 2.51 (3) Hsr Glx 3.17 (3) Pro 2.37 (2) Gly 2.09 (2) Ala 3.19 (3) Val 2.75 (3) Ile Len 2.05 (2) Phe Lys 1.08’ (2) Arg Aec 2.14 (2)

Staphylococcus

aweus peptide (residues 342-348)

0.18

(1) (1) (2) (1)

AL. DISCUSSION

OF RHODOPSIN

(1) (1)

ET

I

Carboxylterminalb

0.14 1.46 1.20 1.07 0.80 0.13 1.78 =1

DOWELL

PEPTIDES

Extradiskal loop” connecting helices V and VI (residues 224253) 1.17 4.35 2.07 + =9 0.79 1.18 6.02 2.96 0.34 1.54 1.46 0.68 0.83

(4) (1) (1) (9) (1) (5) (3) (1) (1) (3) (1)

-

a Peptide CB3 was prepared from pool c of Fig. 3A. Contaminating glycopeptide was removed from pool c by affinity chromatography on Con A-Sepharose (see Methods). Desalting the unbound Con A column fractions on Bio-gel P-2 resulted in pure CB3. *The carboxyl-terminal peptide CB3 was subdigested with S aweus V8 protease. The resulting peptides were separated on Bio-gel P-2. Fractions were pooled on the basis of radioactivity (from the incorporated [“Cjputrescine and amino acid content). Data presented are from an aliquot of this pool. ‘The extradiskal loop peptide connecting helices V and VI was isolated from a mixture of peptides generated by NBS cleavage of pool a in Fig. 3A. Size fractionation of this peptide mixture was performed on Sephadex G-50 in 20% formic acid. Frsctions containing the extradiskal loop peptide were pooled on the basis of radioactivity and amino acid content and the amino acid analysis of the pool was determined. d Numbers in parentheses are the expected composition. Hsr. homoserine; Aec, aminoethylcysteine. e Expected values for lysine analysis are not obtained following reductive methylation.

Gln-Gln-Glu. The digestion mixture was subjected to solid phase sequencing, and the radioactivity released at each step was determined (Table II). These data indicate that all three of the glutamine residues, 236,237, and 238, contain [14C]putrescine in approximately equal amounts. Equal distribution of the 1.95 mol of putrescine among the three sites would result in 0.65 mol putrescine per mole of peptide at each site.

We undertook the study of transglutaminase modification of membrane-bound rhodopsin in order to investigate its topography and function. We sought to locate a site in the rhodopsin sequence modified by Pober et al. (26), and to determine whether other highly exposed glutamine residues might be substrates for transglutaminase. Of particular interest was Gln”, inasmuch as this region of the rhodopsin sequence is highly accessible to proteases [reviewed by Hargrave (9)] and to rhodopsin kinase (11). Since rhodopsin (26) and other protein substrates become crosslinked by the action of transglutaminase on protein-bound glutamines and lysines (8), we felt it necessary to render rhodopsin’s lysines unavailable for this reaction. Any glutamines present as crosslinks within a rhodopsin molecule or to other rhodopsin molecules would reduce the total incorporation of added amine substrate by blocking potential reactive sites. Reductive methylation is a gentle procedure which converts primary amino groups to dimethyl amines with retention of charge, and was our

TABLE

II

RADIOCHEMICAL SEQUENCING OF Staphylococcus ~UT~U.S PROTEASE DIGEST OF HELIX V-VI LOOP PEPTIDE (RESIDUES 224-253)”

?Hl Cycle

counts/min

1 2 3 4 5 6 7 8 9 10

203 101 152 1591 1893 1989 506 204 116 94

Amino acid sequence of residues 233-239 Ala Ala Ala Gln Gin Gin Glu

a The peptide was obtained as shown in Fig. 3B. Its composition is listed in Table I. Subdigestion with SAV8 protease is described under Materials and Methods.

TRANSGLUTAMINASE-MODIFIED

method of choice. We used the procedure of Jentoft and Dearborn (14) with satisfactory results, although a recent procedure of Longstaff and Rando (17) may offer advantages. Our quantitative results indicate complete labeling of rhodopsin’s 10 surface-exposed lysines, whereas the buried retinyl lysine, LYS~‘~, would be unavailable for modification under these conditions. We would also expect complete modification of the ROS membrane aminogroup-containing phospholipids, although we did not attempt to quantify this reaction. Examination of transglutaminasemodified Me2ROS by SDS-PAGE verified that we had been successful in preventing protein oligomer formation. We used transglutaminase from erythrocytes (1) rather than from the more commonly used source, guinea pig liver (8), for reasons of ease and economy of preparation. Our trials with guinea pig liver transglutaminase tend to support the conclusions of Brenner and Wold (l), who found the two enzymes to be extremely similar in their substrate specificity. Since we employed not only a different source of enzyme, but also used a reductively methylated protein substrate, our study cannot be directly compared to that of Pober et al. (26). In both studies the reaction apparently reached a plateau after 6-8 h; however, we found that longer incubation times yield higher incorporation. The maximum incorporation which we obtained-3.5 mol of putrescine into rhodopsin-suggests a minimum of four sites modified. The bulk of the transglutaminase-inserted label is located in the protease-sensitive region connecting helices V and VI, as well as in the protease-sensitive carboxyl-terminal region (although we cannot exclude the possibility of a small percentage of modification at other surface sites). Digestion of transglutaminase-modified rhodopsin by thermolysin, which excises peptide material in these regions, removes 94% of the radioactivity. The CNBr peptide which contains the helix V-VI peptide elutes with the major radioactive peak, and NBS cleavage produces the expected radioactive peptide 224-253. We employed only size fractionation methods in order not to

513

RHODOPSIN

lose minor components, anticipating a heterogeneous modification. Sequence analysis of the subdigested peptide leads to elution of radioactivity in the steps expected for Glnm GlnB7, and Glr?@. Nearly equivalent amounts of radioactivity found at each step suggest that none of the three adjacent residues is a preferred substrate for transglutaminase. Similarly, we have directly demonstrated the modification of Gh?* by isolation of peptides containing it. This residue was modified in 50% of the rhodopsins, which is similar to the approximately 65% degree of occupancy estimated for the other three sites. Results from the studies of rhodopsin topography have allowed construction of a model for the disposition of rhodopsin in its membrane location (13). The results of our current study are in full accord with the body of data on rhodopsin topography and support essential features of the model. Limited proteolysis with trypsin (20,ll) or with thermolysin (16) can be employed to selectively remove the C-terminal region containing Glr?, leaving the remainder of rhodopsin intact. Such a rhodopsin derivative should become modified only in positions G1n236, GlnB7, and Glx?*. It would be of interest to determine if modification of these amino acids has an effect on the interaction of rhodopsin with M, 48,000 protein, G-protein, or rhodopsin kinase. ACKNOWLEDGMENTS We thank Dr. J. Folk for his generous pig transglutaminase which we used for experiments. We also thank Ms. Donna Mr. Walter W. Dimmick for their help procedures involved in the experiments

gift of guinea preliminary Curtis and with various described.

REFERENCES 1. BRENNER,

S. C., AND

Biophgs. 2. BRETT,

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M., AND

J. 211,661-670. 3. BUSTIN, M., AND

(1978)

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J. B. C. (1983)

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522, 74-83. FINDLAY, COLE,

D. R. (1969)

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244,5291-5294. 4. CURTIS, D. R., MCDOWELL, J. H., AND HARGRAVE:, P. A. (1983) Prep. Biochem. 13,83-102. 5. DRATZ, E. A., AND HARGRAVE, P. A. (1983) Trends Biochem. Sci. 8, 128-131.

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6. FAIRBANKS, G., STECK, T. L., AND WALLACH, D. F. H. (1971) Biochemistry 10,2606-2617. 7. FAUCONNET, M., AND ROCHEMONT, J. (1978) Anal. Biochem 91,403-409. 8. FOLK, J. E., AND FINLAYSON, J. S. (1977) A&u. Protein Chewz. 31,1-133. 9. HARGRAVE, P. A. (1982) Prog. Retinal Res. 1, l51. 10. HARGRAVE, P. A., AND FONG, S.-L. (1977) J. Sup-amok Strut. 6,559-570. 11. HARGRAVE, P. A., FONG, S.-L., MCDOWELL, J. H., MAS, M. T., CURTIS, D. R., WANG, J. K., JuszCZAK, E., AND SMITH, D. (1980) Neurochemistry Znt. 1,231-244. 12. HARGRAVE, P. A., MCDOWELL, J. H., CURTIS, D. R., FONG, S.-L., AND JUSZCZAK, E. (1982) in Methods in Enzymology (Packer, L., ed.), Vol. 81, pp. 251-256, Academic Press, New York. 13. HARGRAVE, P. A., MCDOWELL, J. H., CURTIS, D.R.,WANG,J.K.,JUSZCZAK,E.,FONG,S.-L.,MoHANA RAO, J. K., AND ARGOS, P. (1983) Biophys. Strut. Mech 9,235~244. 14. JENTOFT, N., AND DEARBORN, D. G. (1979) J. Biol Chem 254,4359-4365. 15. KUHN, H. (1984) Prog. Retinal Res. 3,123-156. 16. KUHN, H., MOMMERTZ, O., AND HARGRAVE, P. A. (1982) B&him. Biophys. Acta 679,95-100. 17. LONGSTAFF, C., AND RANDO, R. R. (1985) Biochemistry 24,8137-8145.

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