The Cytoplasmic Domain of the H-2Ld Class I Major Histocompatibility Complex Molecule Is Differentially Accessible to Immunological and Biochemical Probes during Transport to the Cell Surface*

An antiserum was generated against a synthetic pep- tide corresponding to a portion of the cytoplasmic domains of the H-2Ld and H-2Db class I major histocompat- ibility complex molecules of the mouse. This antibody preparation, RA, binds exclusively to endoglycosidase H-resistant H-2Ld/Db molecules which are not associated with &microglobulin. Interestingly, acquisition of re- sistance to endoglycosidase H precedes acquisition of R4 reactivity by 30 min. R4-reactive H-2Ld and H-2Db molecules occur on the cell surface and are phosphorylated in vivo. Other studies show that the tyrosine in the cytoplasmic domain is accessible to radioiodination on only a subset of H-2Ld molecules, and that the two-dimensional electrophoretic profiles of phosphorylated H-2Ld/Db molecules, of R4-reactive molecules, and of H-2Ld molecules radiolabeled on this cytoplasmic domain tyrosine are virtually identical. R4-reactive H-2Ld molecules do not undergo the peptide- and p,-micro- globulin-induced conformational changes characteristic of free class I major histocompatibility complex heavy chains. The accessibility of the H-2Ld cytoplasmic domain to R4 and to radioiodination late in biosynthesis and its biological significance are discussed.

Class I major histocompatibility complex (MHCjl molecules are highly polymorphic cell-surface glycoproteins that bind and present endogenously derived antigenic peptides to T lymphocytes (1)(2)(3). The antigen-presenting complex is a noncovalently associated trimolecular assembly consisting of the class I MHC molecule, the P2-microglobulin (&m) light chain, and antigenic peptide (4)(5)(6). Evidence from a number of studies supports a model in which this trimolecular complex is assembled in the endoplasmic reticulum soon after the class I MHC molecule is synthesized (7)(8)(9). The assembled complex is then transported through the Golgi apparatus (10,ll)  Studies on the highly conserved cytoplasmic domain showed that modifications of this region of the H-2Ld molecule of the mouse result in slower transport through the Golgi apparatus (12).2 Hence, it seemed possible that the cytoplasmic domain facilitates the transport of the class I MHC molecule through the cell. To explore this possibility, we generated a polyclonal antibody against a synthetic peptide corresponding to a 13amino acid portion of the cytoplasmic domain of the H-2Ld molecule. The resulting polyclonal antibody, R4, immunoprecipitates H-2Ld and H-2Db class I molecules from cell lysates. Three major features characterize R4-reactive H-2Ld/Db molecules. They are not associated with the p2-m light chain, they are endoglycosidase H (Endo Hbresistant, and some, if not all, are phosphorylated. The latter two characteristics show that R4 binds H-2Ld/Db molecules which have traversed the medial Golgi apparatus. R4-reactive H-2Ld/Db molecules also are found on the cell surface. Hence, it appears that the cytoplasmic domains of the H-2Ld/Db molecules are inaccessible to R4 until late in biosynthesis. In an independent assessment of the accessibility of the H-2Ld cytoplasmic domain, we ascertained whether a tyrosine in this region of the molecule can be radioiodinated. The H-2Ld molecules which undergo radioiodination of the cytoplasmic domain are electrophoretically homogeneous, whereas a more heterogeneous population of H-2Ld molecules is detected by radioiodination of tyrosines on the external domains. The twodimensional gel profiles of H-2Ld molecules which undergo radioiodination on the cytoplasmic domain, those precipitable by R4, and phosphorylated H-2Ld/Db molecules are highly similar. Other studies show that R4-reactive H-2Ld molecules do not associate with exogenously supplied antigenic peptide andor P2-m and do not undergo the peptide-and P2-m-induced conformational changes characteristic of free H-2Ld heavy chains (8,9). We discuss these findings both in terms of potential conformational changes in the H-2Ld/Db cytoplasmic domain (13) and the effects of cellular factors which may bind to and shield the H-2Ld/Db cytoplasmic domains early in biosynthesis.
Synthetic Peptides and Human &-m-The synthetic peptide H-2L13 (GSQSSEMSLRDCK), which is shown enclosed in the box in Fig. 1A shown to bind and be presented by H-2Ld (amino acids 168-176 of murine cytomegalovirus immediate early protein pp89) (17), by (amino acids 345-360 of influenza nucleoprotein, but with a n aminoterminal tyrosine not normally found in this protein) (18), and by H-2Db (amino acids 50-63 of influenza nucleoprotein) (18) were purchased from Applied Biosystems. These peptides are referred to as Ld peptide, Kb peptide, and Db peptide, respectively, in this paper. Human Pz-m was purchased from Sigma and from Calbiochem.
Preparation of Synthetic Peptide Immunogen-The H-2L13 peptide was synthesized and conjugated to keyhole limpet hemocyanin (KLH) using succinimidyl-4-(N-maleimidomethyl)cyclohexane-l-carboxylate (SMCC) as a cross-linker. 60 nmol of KLH in 1 ml of phosphate-buffered saline (PBS), pH 7.5, was incubated with 2.5 pmol of SMCC (dissolved in dimethyl formamide) for 30 min at room temperature. 10 pmol of synthetic peptide in 300 pl of 4 M guanidine hydrochloride (made in PBS, henceforth called Gu-PBS) was brought to pH 3-4 with 17% H,PO,, then brought to 0.15 M dithiothreitol, and incubated for 10 min a t room temperature. The KLH-SMCC and peptide solutions were applied sequentially to a 2-ml Sephadex G-10 column equilibrated with 4 M Gu-PBS, pH 7.5. 200-1.11 fractions were collected. Fractions containing peptide-KLH conjugate were pooled and brought to pH 7.0 with NaOH.
Antibodies-Rabbits were immunized three times over a 2-month period with 100 pg of the H-2L13 peptide-KLH conjugate in complete Freund's adjuvant. Peptide-reactive antibodies were identified by reactivity with H-2L13 conjugated to ovalbumin. The positive antibody preparation thus obtained is called R4 herein. Monoclonal antibodies (mAbs) 30.5.7, 28.14.8, and 64.3.7 have been described previously (8,(19)(20)(21). Subcellular Fractionation-6 x lo7 ELd3 cells were biosynthetically labeled for 90 min a t 100 pCi/ml [35Slmethionine as described below. Cells were then washed once in 15 ml of PBS, once in 4 ml of cavitation buffer (0.245 M sucrose, 10 mM iodoacetamide, 5 mM Hepes, pH 7.0), suspended in 1 ml of cavitation buffer, and loaded into a Parr model 4713 cavitation bomb (Parr Instrument Co., Moline, IL) for nitrogen cavitation (700 p.s.i. of N, for 2 min on ice). The cavitate was centrifuged at 1500 rpm for 5 min to remove the nuclear pellet, and the clarified cavitate was loaded onto the top of a step gradient consisting of 2 g each of 24%, 20%, 16%, 12%, 8%, and 4% (all w/w) Nycodenz (Robbins Scientific Corp., Sunnyvale, CA) dissolved in cavitation buffer. The gradient was centrifuged for 17 h at 4 "C a t 25,000 rpm in a Beckman SW41 swinging bucket rotor, and 0.9-ml fractions were collected, lysed as described below, and subjected to immunoprecipitation analysis.
f?"SIMethionine Labeling of Cells, Immunoprecipitation of Proteins, and Endo H Digestion ofImmunoprecipitate~-[35SlMethionine biosynthetic labeling, pulse-chase studies, double labeling with [35Slmethionine and [32P10,, immunoprecipitation from cell lysates, and Endo H digestions of immunoprecipitates were performed as described previously (12, 16, 22).3 Proteins were eluted from protein A-Sepharose with 0.2% SDS, 0.05 M Tris-HC1, pH 7.5. In the immunoprecipitation experiment shown in Fig. 9, lysates were incubated with the 5 p~ Ld peptide, 5 PM Db peptide, and 0.5 p~ human pz-m a s indicated in the figure legend prior to immunoprecipitation. '2,51-Labeling of Cells, Cavitates, Lysates of Gradient Fractions, and Synthetic Peptides-The cell-surface radioiodination procedure is essentially a modification of a previously published procedure (23). 1.2 x lo7 lymphoid cells per condition were pelleted and washed once with PBS. Radioiodination was carried out at room temperature for 15-30 min in 500 pl of PBS containing 1 uniffml lactoperoxidase, 0.2 uniffml glucose oxidase, 0.5 mCi of NalZ5I (Du Pont-New England Nuclear), and 5 mM p-D-glucose. After three washes with 5 ml of PBS + 5 mM KI, the cells were lysed a s described below. Cell cavitates were radioiodinated by the addition of 1 unit of lactoperoxidase (Sigma), 0.5 unit of glucose oxidase (Sigma), 50 pl of 100 mM p-D-glucose, and 1 mCi of NaIz5I per ml of cavitate. Lysates of gradient fractions (see below) were radioiodinated by the addition of 0.2 unit of lactoperoxidase, 0.1 unit of glucose oxidase, 10 pl of 100 m~ p-D-glucose, and 0.2 mCi of Na125Vml of gradient fraction lysate. The radioiodination of cavitates and lysates was allowed t o proceed a t room temperature for 30 min prior to immunoprecipitation. Synthetic peptides were radioiodinated as described previously." Lysis ofcells and Cell Fractions-Cells were lysed a t a concentration of 2 x lo7 lymphoid cells or 1 x IO7 L cell transfectants per ml of lysis buffer (CHAPS, Boehringer Mannheim, 1 mM phenylmethylsulfonyl fluoride, 0.15 M NaCl, 0.05 M Tris-HC1, pH 7.3, and 10 mM iodoacetamide). Gradient fractions were lysed by the addition of 10 x lysis buffer (5% CHAPS, 0.15 M NaCI, 50 mM Tris-HCI, pH 7.0, and 10 mM phenylmethylsulfonyl fluoride). The final composition of the lysate was 0.22 M sucrose, 4.5 mM Hepes, pH 7.0, 9 mM iodoacetamide, 0.5% CHAPS, 15 mM NaCl, 5 m~ Tris-HC1, pH 7.0, and 1 m~ phenylmethylsulfonyl fluoride.
Co-precipitation Assay for Binding ofpeptide-4 x lo6 HCT-Ld/p2-m cells were lysed, and the lysate was incubated overnight with 5 p~ Ld peptide or Kh peptide which had been radioiodinated a s described above. H-2Ld molecules were then immunoprecipitated with mAb 30.5.7, mAb 28.14.8, or R4. Immunoprecipitates were collected on protein A-Sepharose, and immunoprecipitated proteins were eluted a s described above. Peptide co-precipitating with H-2Ld molecules was determined by counting eluates in a y counter. See Fig. 8 for further details. lo7 ELd3 cells were labeled biosynthetically for 90 min and lysed as described above, and lysates were incubated with 5 p~ Ld peptide, 5 Db peptide, and/or 0.5 PM Pz-m as indicated in Fig. 9. H-2Ld and H-2Db molecules were then immunoprecipitated with mAbs 30.5.7, 28.14.8, 64.3.7, and R4 as indicated in Fig. 9. Class I MHC molecules were eluted as described previously (12) and above. Endo H digestion products were analyzed by SDS-PAGE.
Cyanogen Bromide Digestion-Radiolabeled proteins were purified by transfer to Immobilon membranes (Millipore) and treated with cyanogen bromide (CNBr) as described previously (16). CNBr digestion products were resolved by SDS-PAGE, as described above.

RESULTS
The Anti-cytoplasmic Domain-specific Antibody, R4, Binds the Cytoplasmic Domains of H-2Ld IDb Class I MHC Molecules "Apolyclonal antiserum (R4) against a synthetic peptide identical with 13 amino acids of the mouse H-2Ld cytoplasmic domain ( Fig. lA) was tested for its ability to immunoprecipitate class I MHC molecules. Because the amino acid sequences of the cytoplasmic domains of H-2Ld and H-2Db are identical (25, 26), R4 is expected to bind both of these class I MHC molecules. ELd3 cells (H-2Ld+ and H-2Db+ ) were surface-radioiodinated and lysed, and the lysates were immunoprecipitated with either R4 or with mAb 28.14.8 (which binds the a3 external domains of both H-2Ld and H-2Db (20)). The molecules immunoprecipitated by R4 and mAb 28.14.8 have identical mobilities on SDS-PAGE (Fig. 1B). Further evidence that R4 and mAb 28.14.8 precipitate the same population of molecules is provided by CNBr digestion analysis of the 45-kDa components of the two immunoprecipitates (Fig. X ) .
To investigate its specificity further, R4 was examined for its ability to precipitate H-2Ld molecules having either altered or deleted cytoplasmic domains. In these studies, L cells expressing the wild type H-2Ld molecule (27.5.271, a tail-less H-2Ld molecule (2.2.1.DD5), or an H-2Ld molecule which has undergone deletion of all but seven amino acids of the cytoplasmic domain (911-7 D6), were labeled biosynthetically and immunoprecipitated with R4, mAb 30.5.7, or mAb 28.14.8. The latter two mAbs are external domain-specific, and their binding to H-2Ld is not influenced by the structure of the H-2Ld cytoplasmic domain (12). mAbs 30.5.7 and 28.14.8 both precipitate the tail-less H-2Ld molecule as well as molecules with truncated and full-length cytoplasmic tails (Fig. 2, lanes 2, 3, 5, 6, 8, and 9), while R4 immunoprecipitates only full-length H-2Ld molecules (Fig. 2, lanes l , 4, and 7). RCreactive molecules also are immunoprecipitated from cells expressing H-2Db, but not H-2Ld (data not shown). Taken together with the results shown  (8). To compare the intracellular locations of mAb 64.3.7-reactive and R4reactive molecules directly, biosynthetically labeled ELd3 cells were disrupted by nitrogen cavitation, and subcellular organelles were separated by density centrifugation as described under "Materials and Methods." Class I MHC molecules were H-2Ld - in Fig. 1, these data show that R4 specifically binds to cytoplasmic domain sequences expressed by H-2Ld and H-2Db molecules.

R4-reactive H-2Ld and H-2Dh Molecules Arise Late in Their Biosynthesis and Are Not Associated with the Pz-m Light Chain
"SDS-PAGE analysis of R4 immunoprecipitates from surfaceradioiodinated ELd3 cells (Fig. 1 B ) and overexposure of the autoradiogram in Fig. 2 (data not shown) demonstrate that, unlike mAb 30.5.7 and mAb 28.14.8, R4 binds H-2Ld/Db heavy chains which are not associated with P2-m. Since H-2Ld molecules occur as both P2-m-associated heterodimers and as free heavy chains both early in biosynthesis and at the cell surface (8,12,161, it was of interest to determine when the H-2Ld/Db molecules become R4-reactive. ELd3 cells were pulsed with [3"Slmethionine for 30 min and chased for the times indicated in Fig. 3 prior to immunoprecipitation of H-2Ld/Db molecules with R4, mAb 30.5.7, or mAb 28.14.8. SDS-PAGE resolution of Endo H-digested immunoprecipitates shows that the R4-reactive H-2Ld and H-2Db molecules appear only after a 60-min chase, are entirely Endo H-resistant, and are not associated with P2-m (Fig. 3, lunes [1][2][3][4][5]. In contrast, H-2Ld and H-2Ld/Db class I molecules bound by mAbs 30.5.7 and 28.14.8 occur throughout the various chase periods, exist in both Endo Hsensitive and Endo H-resistant forms, and are associated with P2-m (Fig. 3, lanes 6-15). Approximately 50% of the biosynthetically labeled H-2Ld and H-2Db molecules in ELd3 cells are Endo H-resistant after a 30-min chase period (Fig. 3, lanes 8 and 13 ). However, R4-reactive H-2Ld and H-2Db molecules appear only after a 60-min chase period (Fig. 3, lane 4 ) , and R4 reactivity is greater after 90 min (Fig. 3, lane 5). Thus, H-2Ld/Dh molecules become Endo H-resistant prior to becoming reactive to R4. Similar results were obtained from pulsechase experiments using 27.5.27 L cell transfectants (data not shown).
Not  R4 (lanes 1,4, and 7). mAb 30.5.7 (lanes 2, 5, and 8), or mAb 28.14. 8 (lanes 3, 6, and 9). ELd3 cells were pulsed with I"S1methionine for 15 min, then chased with unlabeled methionine for the periods of time (in minutes) indicated above each lane. At the end of the chase period, the cells were lysed, and lysates were immunoprecipitated with R4 (lanes 1-5). mAb 30.5. 7  (lanes 6-10), or mAb 28.14. 8 (lanes 11-15) H-2 [EflI), and the 12-kDa Pz-m are indicated. immunoprecipitated from gradient fractions with mAb 64.3.7 or with R4, digested with Endo H, and resolved by SDS-PAGE. The vast majority of the mAb 64.3.7-reactive H-ZLd molecules are Endo H-sensitive and occur in denser gradient fractions (Fig. 4A, lanes 5-8). Lesser amounts of mAb 64.3.7-reactive H-2Ld molecules are Endo H-resistant and occur in the less dense fractions of the gradient (Fig. 4A, lanes 2 4 1. In contrast to mAb 64.3.7, R4 precipitates only Endo H-resistant H-2Ld/Db class I molecules which occur in less dense fractions (Fig. 4B). These R4-reactive H-2Ld/Db molecules occur in fractions which include the plasma membrane (data not shown). Similar results have been obtained with cells which express only H-2Ld and not H-2Db (data not shown).
R4 Binds Phosphorylated H-2L"ID" Class I Molecules -Class I MHC molecules undergo phosphorylation in vivo at serine residues in the cytoplasmic domain (25h2 Phosphorylation of class I MHC molecules has been shown to be a postendoplasmic reticulum, and presumably a cell surface, event (27). Since H-2Ld/Dh molecules acquire R4 reactivity late in biosynthesis, we were interested in determining if R4-reactive molecules are phosphorylated in vivo. ELd3 cells were labeled with [35Slmethionine and [32P104 (22), and H-2Ld and H-2Db molecules were precipitated from cell lysates with R4, mAb 30.5.7, or mAb 28.14.8. Immunoprecipitated proteins were digested with Endo H, then resolved by SDS-PAGE under nonreducing conditions. Fig. 5A is an autoradiogram resulting from a direct exposure of the x-ray film. Fig. 5B was generated by placing a piece of paper between the dried gel and the x-ray film, thereby preventing exposure of the film by 35S emissions (22). This analysis shows that all three antibodies precipitate phosphorylated H-2Ld/Db molecules which are Endo H-resistant (compare lanes 2 and 3 of Fig. 5A to lanes 2 and 3 of Fig.  5B ). Since mAb 30.5.7 precipitates exclusively P2-m-associated H-2Ld molecules from P2-m+ cells (28,29), and since mAb 30.5.7-reactive H-2Ld molecules are phosphorylated (Fig. 5B, lane 2), phosphorylation of the H-2Ld heavy chain does not require loss of P2-m. In addition to phosphorylated H-2Ld/Db heavy chains, mAb 28.14.8 and R4 also bind phosphorylated H-2Ld/Db heavy chain dimers (Fig. 5B, lanes 1 and 3 ) , which have been shown to arise late in biosynthesis in P2-m+ cells (16).
"SP2P-Labeled proteins immunoprecipitated by mAbs 30.5.7 and 28.14.8 and by R4 were analyzed further by twodimensional gel electrophoresis. (Heavy chain dimers are not seen on these gels because the two-dimensional gels were run   under reducing conditions.) H-2Ld molecules precipitated from 35S/32P-labeled ELd3 cells by mAb 30.5.7 (Fig. 6A) display marked heterogeneity. However, the autoradiogram generated by placing a piece of paper between the dried gel and the x-ray film shows that only a subset of these molecules is phosphorylated (Fig. 6B). Indeed, mAb 30.5.7-precipitable 32P-labeled H-2Ld focuses to a single spot (Fig. 6B ). Similarly, 32P-labeled H-2Ld/Db molecules bound by mAb 28.14.8 and by R4 are electrophoretically homogeneous (Fig. 6, C and D, respectively). However, the two-dimensional profile of R4-reactive 35S/32Plabeled H-2Ld/Db is identical whether or not the 35S emissions are blocked by a sheet of paper (data not shown). Taken together, the data in Figs. 5 and 6 confirm that R4-reactive molecules are Endo H-resistant and show that R4-reactive molecules include the homogeneous subset of H-2Ld/Db molecules which is phosphorylated in vivo.

emissions. Mobilities of Endo Hresistant H-2Ld/D') dimer (H-2 Dimer [EHr]), Endo H-resistant H-2Ld and H-2Ld/DL' heavy chains (H-2 /El?]), Endo H-cleaved H-2L" and H-2Ld/D" heavy chains (H-2 /EHrl
The Cytoplasmic Domain &rosine of Only a Subset of H-2Ld Molecules Is Accessible to Radioiodination-We next used radioiodination of membrane vesicles to determine if the inaccessibility of Endo H-sensitive H-2Ld molecules to R4 is unique to this probe or reflects a general inaccessibility of the cytoplasmic tail. Membranous vesicles generated by gentle cavitation of ELd3 cells were either radioiodinated and lysed or lysed and radioiodinated. H-2Ld molecules then were precipitated with mAb 30.5.7. Under the cavitation conditions used, the external domains of class I molecules remain inside the lumena of the vesicles generated by cavitation and thus are protected from radioiodination, while their cytoplasmic domains extrude from the vesicles and are available to radioiodination (Fig. 7A). Proof that the orientation of cellular membranous components is maintained in the cavitates is provided by the fact that the p2-m co-precipitated with H-2Ld molecules is not radioiodinated (Fig. 7C). In contrast, radiolabeled P2-m occurs in mAb 30.5.7 immunoprecipitates of H-2Ld molecules from radioiodinated lysates (Fig. 7B 1  analysis reveals that the H-2Ld molecules radioiodinated in cell lysates display electrophoretic heterogeneity (Fig. 7B ), similar to that which is observed for H-2Ld molecules labeled in vivo (Fig. 6A). In contrast, H-2Ld molecules radioiodinated in cell cavitates are electrophoretically homogeneous (Fig. 7C). That the greater heterogeneity of the sample shown in Fig. 7B is not due to oxidation or multiple radioiodination events was demonstrated in control experiments in which purified H-2Ld molecules which were previously radioiodinated in cell cavitates were subsequently iodinated with nonradioactive iodine. These molecules also focus into a single spot with an indistinguishable PI from that of "cavitate" radioiodinated H-2Ld (data not shown). The two-dimensional gel electrophoretic mobility of the single spot observed in mAb 30.5.7 immunoprecipitates of radioiodinated cavitates (Fig. 7C) is virtually identical with that of the prominent "P-labeled spot in mAb 30.5.7, mAb 28.14.8, and R4 immunoprecipitates from "P-labeled samples (Fig. 6, B-D, and data not shown). R4-reactive H-2LdlDh Molecules Do Not Associate with Peptide a n d Pz-m in Vitr-The lack of association of R4-reactive H-2Ld/Db heavy chains with P2-m implies that these molecules also are not associated with antigenic peptide. The ability of R4-reactive H-2Ld molecules to associate with antigenic peptide was assessed in a co-precipitation assay (described under "Materials and Methods") using HCT-LdIP2-m cells, which express H-2Ld and mouse P2-m. HCT-LdIP2-m cell lysates were incubated overnight with radioiodinated Ld peptide or radioiodinated Kh peptide, and H-2Ld molecules then were immunoprecipitated with R4, mAb 30.5.7, or mAb 28.14.8. mAbs 30.5.7 and 28.14.8 each co-precipitate significantly more lzsI-Ld peptide than 12sI-Kh peptide from HCT-LdIP2-m cell lysates (Fig. 8,   panels 1 and 2, respectively). In contrast, R4 co-precipitates very little of either peptide and, in fact, less Ld peptide than Kb MHC Cytoplasmic Domain 21267 peptide from these cell lysates (Fig. 8, panel 3 ) . Furthermore, precipitation of H-2Ld molecules by R4 does not deplete Ld peptide-binding H-2Ld molecules reactive with mAb 30.5.7 (Fig. 8, panel 4).
Based on their studies of the cytoplasmic domain of H-2Kb, Smith and Barber (13) suggested that dissociation of a class I MHC heavy chain from P2-m induces a conformational change in the cytoplasmic domain, rendering it accessible to antibodies specific for exon 7-encoded sequences. Other studies have shown that addition of antigenic peptide and P2-m induces a conformational change in the class I MHC molecule (8,18,(28)(29)(30)(31). Of particular interest here was the demonstration by Hansen and colleagues ( 8 ) that addition of peptide to cell lysates results in a depletion of Endo H-sensitive, mAb 64.3.7reactive H-2Ld molecules and a concomitant increase in Endo H-sensitive, mAb 30.5.7-reactive H-2Ld molecules. To determine if R4-reactive H-2Ld molecules could be similarly affected by specific peptide and P2-m, ELd3 cells were labeled biosynthetically for 2 h and lysed, and the lysates were supplemented with the peptide and P2-m additions indicated in the legend to  Fig. 9, lanes 1-4) and mAb 28.14.8 (H-2Ld and H-2Db, Fig. 9, lunes 5-6). Addition of Ld peptide and P2-m to cell lysates prior to immunoprecipitation causes an increase in Endo Hsensitive H-2Ld in mAb 30.5.7 immunoprecipitates and a decrease in Endo H-sensitive H-2Ld in mAb 64.3.7 immunoprecipitates, but does not affect the Endo H-resistant H-2Ld (Fig.  9, lanes 2 and 4). Similarly, addition of Ld peptide, Dh peptide, and P2-m to cell lysates results in an increase of Endo Hsensitive H-2Ld/Dh precipitated by mAb 28.14.8, again without affecting the Endo H-resistant H-2Ld/Db molecules (Fig. 9, lane 6). Only Endo H-resistant H-2Ld is observed in R4 immunoprecipitates, and its quantity is unchanged by the addition of Ld peptide, Db peptide, and Pz-m (Fig. 9, lanes 7 and 8). These results show that H-2Ld/Dh molecules bound by R4 are unable to bind peptide and to undergo a peptide-and P2-m-induced conformational change in vitro. DISCUSSION In this paper we have examined the accessibility of the cytoplasmic domain of H-2Ld and H-2Dh class I MHC molecules to immunological and biochemical probes. We show that the cytoplasmic domain-specific antibody R4 detects H-2Ld/Db molecules which are not associated with P2-m. Previous studies showed that a relatively large pool of free H-2Ld h e a w chains occurs in P2-m+ cells (8). However, R4 immunoprecipitates only a small proportion of the free H-2Ld heavy chains in a cell.
Cellular Location and Genesis of R4-reactive H-2LdIDh Molecules"R4-reactive molecules are characteristically Endo Hresistant and electrophoretically homogeneous. Consistent with a postmedial Golgi localization, R4-reactive H-2Ld/Db class I molecules occur in less dense fractions (which include the plasma membrane) on density gradients. R4-reactive H-2Ld/Db molecules occurring at the cell surface exist both as free heavy chains and as heavy chain dimers. While H-2Ld and H-2Db have been shown to be transported to the cell surface in a p2-m-form, we believe that RCreactive molecules arise from H-2Ld-P2-m heterodimers after the dissociation of P2-m. This conclusion stems from two observations. First, R4-reactive molecules arise late in the biosynthesis of H-2Ld even though free H-2Ld heavy chains occur early in biosynthesis. Secondly, H-2Ld molecules synthesized in P2-m-cells do not become phosphorylated even though they are expressed at the cell surface.2 Since both R4-reactive free H-2Ld heavy chains and p2-m-associated (mAb 30.5.7-reactive) H-2Ld molecules are phosphorylated, R4-reactive H-2Ld molecules may arise from mAb 30.5.7-reactive H-2Ld molecules which have dissociated from peptide and Pn-m. H-2Ld molecules are likely to become R4reactive at the cell surface since there is a delay between acquisition of resistance to Endo H by H-2Ld molecules and their reactivity to R4 (as shown by comparison of R4 and mAb 30.5.71 mAb 28.14.8 immunoprecipitates in Fig. 3).
The Nature of the Cytoplasmic Domain Inaccessibility-A highly conserved tyrosine occurs in the cytoplasmic domains of most class I MHC molecules (26). This tyrosine is separated from the epitope recognized by R4 (peptide H-13) by only four amino acids (Fig. lA). The studies shown in Fig. 7C demonstrate that when membrane vesicle preparations are radioiodinated only a subset of H-2Ld molecules is labeled at the cytoplasmic domain tyrosine. The two-dimensional gel profile of these molecules is similar to that of R4-reactive H-2Ld molecules ( Fig. 6 and data not shown).
bind exogenous peptide because they already are occupied by peptide. However, in other studies, we have found that free H-2Ld heavy chains do bind peptide, albeit poorly, and that the bound peptide exchanges r e a d i l~.~ While by no means conclusive, these results are inconsistent with the idea that the conformation of the H-2Ld/Db cytoplasmic domain is altered by the association of the external domains with peptide and Pn-m. Another argument against this particular conformational change model is the finding that only a small fraction of free H-2Ld heavy chains in a cell is R4-reactive (Figs. 4 and 9h4 The amount of R4-reactive H-2Ld/Db does not increase during a prolonged label.2 It is therefore tempting to speculate that R4 reactivity occurs soon before degradation of dysfunctional H-2Ld/Db molecules. Ongoing studies in our laboratory are directed toward investigation of this possibility. Phosphorylation of the cytoplasmic domain serines on the H-2Ld molecule might also influence R4 reactivity, especially since the immunogenic peptide bound by R4 includes serines which potentially can be phosphorylated (251, and some, if not all, R4-reactive H-2Ld molecules are phosphorylated. Because the immunogenic peptide was not phosphorylated prior to immunization, it is unlikely that phosphorylation per se determines R4 reactivity. However, phosphorylation of the cytoplasmic domain could influence R4 binding by affecting the conformation of the cytoplasmic domain or altering interactions of the cytoplasmic domain with other proteins. An obvious way to distinguish between these possibilities is to determine the R4 reactivity of native and denatured H-2Ld/Db molecules. Accordingly, Western blot analysis of R4-reactive molecules is in progress.
Implications for Intracellular Thansport of Class I MHC Molecules and Other Integral Membrane Proteins-The inaccessibility of the cytoplasmic domain of the H-2Ld molecule to antibody probes and radioiodination until late in biosynthesis is reminiscent of the reduced accessibility of the cytoplasmic domain of vesicular stomatitis virus G protein to proteases early in biosynthesis (32). These observations are interesting in light of previous studies which demonstrated that H-2Ld molecules with full-length cytoplasmic domains are transported through the cell more rapidly than are H-2Ld molecules with truncated cytoplasmic tails (12). The facilitation of intracellular transport by the cytoplasmic domain is not unique to H-2Ld; in fact, it has been reported for a number of integral membrane proteins (33)(34)(35)(36)(37). However, there is no similarity in cytoplasmic tail length or sequence of the various proteins examined in these studies (12, [32][33][34][35][36][37]. It is tempting to propose that facilitation of intracellular transport by the cytoplasmic domains of integral membrane proteins destined for the cell surface is due to their association with intracellular components and that cytoplasmic domain accessibility to biochemical and immunological probes late in biosynthesis is due to dissociation from these components. Ongoing studies are directed at determining the relative contributions of conformation, phosphorylation, and association with cellular proteins to differential cytoplasmic domain accessibility during intracellular transport.