Traffic control of completely assembled MHC class I molecules beyond the endoplasmic reticulum1

J. Mol. Biol. (1997) 266, 993±1001

Traffic Control of Completely Assembled MHC Class I Molecules Beyond the Endoplasmic Reticulum Sebastian Joyce Department of Microbiology & Immunology, The Pennsylvania State University College of Medicine, Milton S. Hershey Medical Center, Hershey, PA 17033, USA

It is generally assumed that MHC class I molecules arrive at the plasma membrane following biosynthesis, assembly and architectural editing in the endoplasmic reticulum by constitutive forward movement without requirement for speci®c signals (bulk ¯ow). If this is true then all overexpressed completely assembled class I molecules should arrive at the cell surface. To study the itinerary of class I traf®c beyond the endoplasmic reticulum, mammalian cells that overexpress 20 to 50-fold higher amounts of the constituent heavy and light chains were established. Thorough biochemical analyses revealed that such overexpressed molecules assemble with authentic peptides that contain the canonical class I binding anchor motif in almost 1:1 stoichiometry and impart thermal stability to the heterotrimeric complex. Despite complete assembly, however, only a fraction of the overexpressed molecules reaches the cell surface. Almost all of the overexpressed class I molecules are sialylated, thus traf®c as far as the trans-Golgi or the trans-Golgi network. Overexpression of class I molecules do not seem to cause a ``traf®c jam'' in the exocytic pathway because the kinetics of traf®c of Sindbis virus structural proteins to the plasma membrane are almost identical when comparing the nonengineered and engineered cells. Thus the steady state expression of class I molecules at the cell surface is further controlled either in the Golgi apparatus or at the plasma membrane. # 1997 Academic Press Limited

Keywords: MHC class I molecules; class I assembly; traf®c control; protein targeting; Golgi apparatus

Introduction Major histocompatibility complex (MHC) encoded class I molecules control the development of CD8‡ T cells and the induction of self tolerance in the thymus, immunesurveillence against cytosolic pathogens and tumors as well as the acceptance or rejection of allografts (Zinkernagel & Doherty, 1979). The control of these T cell processes requires the chaperoning of short peptides, eight to ten amino acid residues long, to and their presentation at the cell surface by class I molecules. In normal cells, it is estimated that class I molecules continuously bind and present over 10,000 different peptides derived from self proteins (Joyce & Abbreviations used: MHC, major histocompatibility complex; ER, endoplasmic reticulum; mAb, monoclonal antibody; PTH, pheaylthiohydantoin; TCA, trichloroacetic acid; PBS, phosphate-buffered saline; FCS, foetal calf serum; TFA, tru¯uoro acetic acid; IEF, isoelectric focussing. 0022±2836/97/100993±09 $25.00/0/mb960822

Nathenson, 1996). Recognition of non-self peptides derived from cytosolic pathogens so presented triggers CD8‡ T cells to lyse the infected cells (Townsend & Bodmer, 1989). Thus class I molecules control both the nature (i.e. which peptides are presented) and quantitative (i.e. how many copies of a single peptide class I complex per cell) aspects of peptide antigen presentation. Since they are type I integral membrane glycoproteins, class I heavy chains assemble with b2-microglobulin (b2-m) and peptides in the endoplasmic reticulum (ER). MHC class I molecules assemble and achieve the native conformation, as judged by acquisition of conformation dependent antibody epitopes, within 2 to 15 minutes of synthesis (Levy et al., 1991; Neefjes et al., 1993; Joyce & Nathenson, 1996). However, the halftime (t1/2) taken by different class I proteins to egress from the ER, vary between t1/2 of 20 minutes for H-2Kb and Dd and 55 minutes for H-2Kd, Db and Ld (Degan & Williams, 1991; Jackson et al., 1994). The lag period between complete assembly # 1997 Academic Press Limited

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Traf®c Control Beyond the Endoplasmic Reticulum

and the egress of class I proteins from the ER suggest speci®c retention of these molecules, probably for architectural editing prior to their translocation to the proximal Golgi stack. Upon leaving the ER, it is generally assumed that class I molecules rapidly arrive at the cell surface (Jackson et al., 1994). However, cogent data for this assumption is lacking.

Results Majority of the overexpressed class I molecules are not expressed at the cell surface An approach to study the late events in the transport of MHC class I would be to overexpress completely assembled molecules so that they would saturate a rate limiting step(s), if there is one, during their traf®c to the plasma membrane. Thus, mammalian cell lines that overexpress H-2Kb (Kbhigh) and Db (Db-high) were generated (see Methods). To compare the steady state levels of H2Kb and Db molecules in the high expressor cell lines and the commonly used H-2b class I positive cell lines, 5  105 of each engineered line and 5  106 EL-4 cells, were biosynthetically labeled with [35S]methionine, lysed and immunoprecipitated with conformation dependent monoclonal antibodies (mAb), Y3 (EL-4 and Kb overexpressors) and B22-249 (EL-4 and Db overexpressors). Comparison of the ®ve Kb-high cells with those of EL-4 revealed that the engineered cell lines expressed between ®ve and 20-fold more of Kb molecules than EL-4 cells (Figure 1A left panel and B). Similarly, Db-high cells express between ten and 50fold more of Db compared to EL-4 cells (Figure 1A right panel and B). Further, comparison of class I expression in Kb-high, Db-high and RMA cells yielded similar results (data not shown). This difference in the levels of class I synthesized is not due to differential metabolic labeling of cells (see Figure 2E). Thus, the engineered NS0 cells overexpress between ®ve and 50-fold more of H-2Kb and Db compared to EL-4 and RMA. Interestingly, however, ¯ow cytometric analysis of cell surface H-2Kb and Db expression on Kb-high and Db-high cells using Y3 and B22-249, respectively, revealed that they express approximately the same levels of class I molecules as do EL-4 (Figure 1C) and RMA (data not shown). This was consistently seen in all experiments. Thus, between 80% (Kb-high 5) and 98% (Db-high 2) of the overexpressed class I molecules are not stably expressed at the cell surface. Overexpressed class I molecules are completely assembled The overexpressed class I molecules may not be negotiating the secretory pathway because of incomplete assembly with b2-m and/or peptides. Such incompletely assembled class I molecules are retained in the ER (Degan & Williams, 1991; Hsu et al., 1991; Jackson et al., 1994). Although NS0 cells

Figure 1. Cell surface H-2Kb and Db expression remains almost the same despite ®ve to 50-fold higher levels of biosynthesis of class I heavy and light chains. One hour [35S]methionine labeled cell lysates of 5  105 Kb-high (clones 1 to 6) and Db-high (clones 1 to 3), together with a tenfold excess of an EL-4 cell lysate were immunoprecipitated with conformation-dependent mAb speci®c for H-2Kb (Y3) and Db (B22-249), separated by SDS-15% PAGE and visualized by autoradiography (A). The heavy chain band in the autoradiogram shown in A was quantitated by scanning densitometry and represented in arbitrary units, as the product of the scanned intensity and the difference in the cell number (which is equal to 10) (B). The steady state cell surface expression of H-2Kb and Db molecules in non-engineered EL-4 and overexpressor cells is almost the same (C). EL-4 and overexpressor cells were reacted with Y3 and B22-249, stained with ¯uorescin-isothiocyanate conjugated antimouse immunoglobulin and detected by ¯ow cytometry. H, heavy chain; L, light chain (b2-m).

were engineered to co-express the heavy and light chain cDNA from the same plasmid construct, it is possible that the light chain is not expressed as ef®ciently as the heavy chain. To determine the availability of b2-m for class I assembly, post nuclear lysates of [35S]methionine-labeled Kb-high-5 and Db-high-2 cells (henceforth called Kb-high and Db-high, respectively), were successively immuno-

Traf®c Control Beyond the Endoplasmic Reticulum

995 precipitated with conformation-dependent antibodies (Kb: Y3 and Db: B22-249) and conformationindependent antibodies (Kb: anti-X8, an antiserum against the cytoplasmic tail region of H-2K molecules (Joyce et al., 1994a) and Db: 28-14-8s), followed by immunoprecipitation with an antiserum against b2-ma. Both Kb-high and Db-high contain free b2-m within the cells and in the culture supernatant (Figure 2A). Hence, b2-m is available in suf®cient amounts to support ef®cient assembly of overexpressed class I molecules. To determine if the overexpressed class I molecules are complexed with self peptides, Kb-high, Dbhigh and EL-4 cells were biosynthetically labeled with [3H]tyrosine. [3H]Tyrosine was used as the label because tyrosine is a residue utilized by Kb and Db binding self peptides (Van Bleek & Nathenson, 1991) and is also present in the heavy and light chains. This would then allow the quantitation of the amount of [3H]tyrosine in the af®nity puri®ed class I complex (heavy and light chains and peptides), the Centricon 3 ®ltrate and the reversed-phase HPLC (RP-HPLC) puri®ed peptides, by scintillation counting, from which a comparison can be made between those molecules expressed by the engineered cells and EL-4 cells (Figure 2B to D). Such a comparison of Y3 af®nity puri®ed molecules revealed that Kb-high cells express 35 times the amount of Kb expressed by EL-4 cells (Figure 2B). This difference in the levels of class I molecules synthesized is not due to differential metabolic labeling of cells (Figure 2E). Heat-denaturation of Kb at low pH followed by separation of the low-molecular-mass fraction by Centricon 3 ®ltration, showed that 30 fold more of the self peptides can be isolated from the overexpressed Kb than from those expressed by EL-4 (Figure 2C). Quantitation of RP-HPLC fractionated Centricon 3 ®ltrate revealed that 20-fold more of self peptides

Figure 2. Overexpressed H-2Kb and Db class I molecules completely assemble with high af®nity self peptides. A, b2-Microglobulin is in excess to support the assembly of overexpressed class I Kb and Db heavy chains. Kbhigh and Db-high cells were steady-state labeled and sequentially immunoprecipitated in the order described: Kb-high lysate: 28-14-8S (a control mAb that recognizes b2-m free and associated Db and Ld heavy chains), Y3 (conformation dependent anti-Kb), anti-X8 (conformation independent H-2K locus speci®c rabbit heteroantiserum) and anti-b2-m (rabbit heteroantiserum reactive against b2-ma); Db-high lysate: anti-X8 (control), B22-249 (conformation dependent anti-Db), 28-14-8S and anti-b2-m. The supernatant was also sequentially immunoprecipitated: Kb-high supernatant: anti-X8 followed by anti-b2-m; Db-high supernatant: 28-14-8S followed by anti-b2-m. B to E. Overexpressed H-2Kb and Db molecules associate with self peptides in almost unimolar ratio. Self peptides associated with af®nity puri®ed H-2Kb or Db expressed by EL-4, Kb-high and Db-high cells biosynthetically labeled with [3H]tyrosine (an amino acid incorporated into the heavy and light chains and peptides) were isolated and fractionated by reversed-phase HPLC. An aliquot of each component, the class I (B) and associated

self peptides separated into the Centricon 3 ®ltrate (C) and reversed-phase HPLC puri®ed (D), was quantitated by scintillation counting. The amount of radiolabel incorporated into the cells was quantitated by monitoring an aliquot of the post nuclear lysate (E). The data represent the average and the standard error of the mean of three experiments. (F) Overexpressed H-2Kb are associated with high af®nity self peptides and impart thermostablity to the molecule. Post-nuclear detergent lysates of [35S]methionine labeled EL-4 and Kb-high cells were divided in equal aliquots and each aliquot was incubated at 4 C, 20 C, 37 C, 42 C or 56 C for 15 minutes and immunoprecipitated with Y3. The immune precipitates were resolved by SDS-PAGE, visualized by autoradiography and quanti®ed by densitometric scanning of the b2-m band that co-precipitated with the heavy chain. The intensity of b2-m band (in arbitrary units) co-precipitated at 4 C was taken as 100%. The amount of light chain co-precipitated at different temperatures was related to that at 4 C in percentage (y-axis: Kb stability) and plotted against the inverse of temperature in Kelvin (x-axis).

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Traf®c Control Beyond the Endoplasmic Reticulum

Table 1. The binding motif of peptides assembled with overexpressed b2-m associated H-2Db molecules Form of class I Native, b2-m‡

mAb (speci®city) B22-249 (a1)

1

2

3

4

5

Aa F Y I

A Q M G

P I Y L V F

R E H Q K T S V

Nb

Position in peptide 6 7 K I Q F L V

H E T

8

9

10

11

12

H Y K T

M I L

±c

±

±

a

The assignment of residues at a given position (cycle) in the peptide is based on the decreasing order of their yields. Residues in boldface type indicate the Db binding anchor motif (Falk et al., 1991). Yields of phenylthiohydantoin (PTH) derivatized amino acids signi®cantly decreased beyond the tenth Edman degradation cycle to barely detectable levels. b c

are associated with overexpressed Kb than those expressed by EL-4 (Figure 2D). The reduction in the yield of peptides is due to losses incurred during the puri®cation steps (Jardetzky et al., 1991; Joyce et al., 1994b). A similar comparison of Db and its associated self peptides revealed that Db-high cells express 35-fold more of the class I molecule and 30-fold and 24-fold more of Centricon 3 and RP-HPLC fractionated self peptides, respectively, when compared to those expressed by EL-4 cells (Figure 2B to D). Thus the overexpressed class I molecules assemble with self peptides in close to 1:1 stoichiometry. Further, the data also suggest that cells generate at least a 20-fold excess of self peptides than is required to support the assembly of class I molecules in non-engineered cells. Although almost all of the overexpressed class I molecules are associated with self peptides, it is possible that the large majority of these peptides are not authentic (shorter or longer than the canonical class I binding peptides and/or lack one or more of the anchor, dominant or accessory, residues). Such peptides do not bind class I molecules with suf®ciently high af®nity to pass architectural editing in the ER. Therefore, the overexpressed class I molecules inef®ciently translocate to the cell surface. Thermal stability of class I molecules is a function of high af®nity binding of authentic peptides that can be measured by the amount of b2-m co-immunoprecipitating with the heavy chain at various temperatures (Jackson et al., 1992). Cells were metabolically labeled with [35S]methionine for 30 minutes and equal aliquots of the post nuclear fraction were incubated at 4, 20, 37, 42, and 56 C for 15 minutes. Following temperature treatment, the lysates were immunoprecipitated with conformation-dependent mAb, Y3 or B22-249. The amount of b2-m co-precipitating with the heavy chain at different temperatures was quanti®ed by densitometry following SDS-PAGE and autoradiography. This feature of the overexpressed class I molecule was compared to class I molecules expressed by EL-4 under similar conditions of temperature. The data clearly indicate that all the overexpressed class I molecules are as thermostable, if not more so, as those assembled in normal cells

(Figure 2F). Similar results were obtained with overexpressed Db molecules (data not shown). Thus, the peptides associated with the overexpressed class I molecules are assembled with high af®nity interactions between the component chains. Class I molecules can bind 2000 to over 10,000 different self peptides. Class I allele speci®c peptide binding is accomplished by structurally similar residues, a combination of which is called the class I peptide binding motif (Falk et al., 1991). The large majority of the self peptides associated with the overexpressed class I molecules might have a binding motif that is different from the hitherto fore reported motif. This could then affect the structure of the class I molecule and hence its intracellular traf®c and cell surface expression. If the canonical binding motif is maintained within all self peptides assembled with overexpressed class I, then amino acid sequence analysis of the total peptides isolated from them will conform to the already reported motif. If the binding motif is maintained only within self peptides that are expressed at the cell surface (2 to 20%) then the reported motif will be barely or will not at all be detectable by sequence analysis of the peptides. To determine the binding motif within the overexpressed Db associated self peptides, the molecules were isolated by Centricon 3 ®ltration of denatured af®nity puri®ed Db molecules expressed by 5  109 Db-high cells. Edman degradation of the concentrated Centricon 3 ®ltrate revealed that almost all of the associated peptides are short, nine amino acid residues long (Table 1). Further, the reported motif consisting of asparagine at position 5 and carboxy-terminal methionine, isoleucine or leucine (Falk et al., 1991) is maintained within the self peptides associated with the overexpressed Db molecules (Table 1). We previously reported that the pool sequence analysis of self peptides associated with overexpressed Kb molecules maintained the canonical Kb binding motif (Joyce et al., 1994a). Thus, from the results presented in Figure 2 and Table 1, I conclude that by all testable biochemical criteria the overexpressed H-2Kb and Db molecules are completely assembled; i.e. they assemble as thermostable tri-

Traf®c Control Beyond the Endoplasmic Reticulum

997

Figure 3. H-2Kb and Db molecules of the overexpressor cell lines negotiate the secretory pathway with kinetics similar to class I expressed by non-engineered cells while maintaining almost normal traf®c through the exocytic pathway. A, Kinetics of class I egress from the endoplasmic reticulum. Cells were pulse labeled for ten minutes and chased for the indicated periods of time. Post-nuclear lysates were immunoprecipitated with the respective mAb, Y3 (anti-Kb) and B22-249 (anti-Db). The immune complexes were eluted, divided in two equal aliquots and either mock (ÿ) or endoglycosidase H digested, trichloroacetic acid (TCA)-precipitated and processed for SDS-PAGE and autoradiography. The differences in the intensities of the bands in the different lanes re¯ect uneven precipitation of the heavy chain by TCA. This was corrected by direct digestion of the immune complexes without elution and TCA precipitation (see Figure 3C). Endo H: endoglycosidase H; r: Endo H resistant; s: Endo H sensitive. B, Overexpressed H-2b class I molecules are sialylated. Kb-high and Db-high cells were steady-state labeled (30 minutes; S) or pulse labeled (ten minutes) and chased for the indicated periods of time. Post-nuclear lysates were immunoprecipitated with Y3 or B22-249, resolved by one-dimensional vertical gel isoelectric focusing (IEF) and detected by autoradiography. H-2Kb and Db heavy chains have one and two glycosylation sites, respectively, and hence have three and six sialic acid residues added on during ®nal maturation in the trans-Golgi or the trans-Golgi network. Each sialic acid residue adds one negative charge to the protein thereby lowering its pI (isoelectric point). The change in pI is detected by IEF. C, Overexpressed class I molecules turn over in a low pH compartment. Kb-high and Db-high cells were pulselabeled for ten minutes and chased for the indicated time periods either at 37 C, 20 C or in the presence of ammonium chloride at 37 C. At the end of the chase, class I molecules were immunoprecipitated with Y3 or B22-249; the immune complexes were directly digested with endoglycosidase H, and processed for SDS-PAGE and autoradiography. D and E, Unhindered traf®c of Sindbis virus structural proteins in the overexpressor cells. NS0, (parent cell line) in which the overexpressors were generated, Kb-high and Db-high cells were infected with Sindbis virus for 16 hours, metabolically labeled for ten minutes and chased for the indicated periods. Post-nuclear lysates were sequentially immunoprecipitated, ®rst with E1 speci®c then with pE2 ‡ E2 speci®c rabbit heteroantiserum and processed for SDS-PAGE and autoradiography (D). The maturation of E2 occurs in transit from the trans-Golgi to the cell surface from its precursor, pE2, by proteolytic cleavage. Sindbis virus was isolated by ultracentrifugation of the labeling supernatant at the end of the various chase times, extracted with 0.5% SDS and the integral membrane glycoproteins solubilized in 0.5% Nonidet P-40. Viral E1 glycoprotein was immunoprecipitated from the virus that bud into the labeling supernatant and processed as described above (E). In this experiment, [35S]methionine/[35S]cysteine incorporation was different in the three cell lines. However, the ratio of the structural proteins synthesized to the mature form (E2; D) or the secreted molecule (E1; E) is approximately the same. Note that the synthesis and traf®c of Kb and Db in the overexpressor cells are not affected by Sindbis virus infection (data not shown).

998 molecular complexes of heavy and light chains and self peptides, containing the canonical binding motif, in close to unimolar ratio. Overexpressed class I molecules traffic as far as the late Golgi compartment Given that the overexpressed class I molecules are completely assembled, how far do they traf®c en route to the cell surface? To address this question, EL-4, Kb-high and Db-high cells were pulse labeled for ten to 15 minutes, chased for varying periods of time in the presence of excess cold methionine and prepared for immunoprecipitation with Y3 and B22-249. The immune precipitates were divided in two equal aliquots; one part was digested with endoglycosidase H and the second was incubated in the digestion buffer lacking the enzyme. Glycoproteins attain endoglycosidase H resistance upon egress from the ER and arrival into the proximal Golgi stacks (cis- and/or medial-Golgi; Kornfeld & Kornfeld, 1985). Thus Kb and Db molecules egress from the ER at rates similar to that of the non-engineered EL-4 cells (Figure 3A) which almost matches the rate previously reported for these molecules (t12 of 20 minutes for Kb and 55 minutes for Db; Degan & Williams, 1991). Addition of three sialic acid residues per glycosyl group is the terminal sugar modi®cation of class I glycoproteins that occurs in the trans-Golgi and/or the trans-Golgi network (Ljunggren et al., 1990; Neefjes et al., 1990). Overexpressed Kb and Db were immunoprecipitated from pulsed and chased Kb-high and Db-high cells to determine how far into the Golgi apparatus they traf®c. The immune complexes were then resolved by one-dimensional isoelectric focusing, a method that resolves proteins by charge. As shown, almost all of the overexpressed class I molecules are sialylated (Figure 3B) at a rate similar to that established for these molecules in non-engineered cell lines as well as in mitogen activated lymphocytes (Van Kaer et al., 1992; Machold et al., 1995). Taken together, the data indicate that the overexpressed class I molecules are not retained in the ER but traf®c as far as the transGolgi or the trans-Golgi network. To determine whether overexpressed class I molecules are retained in the post-ER compartment or degraded, Kb-high and Db-high cells were pulseradiolabeled for ten to 15 minutes and chased for the indicated periods of time either in the absence or presence of ammonium chloride at 37 C (Figure 3C). At 37 C, class I molecules are degraded with time (Figure 3C). The half lives of overexpressed Kb and Db is similar to those reported for these molecules expressed at endogenous levels in cell lines and lymphocytes (Van Kaer et al., 1992; Machold et al., 1995). The presence of ammonium chloride, an agent that disrupts the functioning of acidic vesicles, rescues overexpressed class I molecules from degradation (Figure 3C). Similar results were obtained with chloroquine, a lysosomotropic agent that also af-

Traf®c Control Beyond the Endoplasmic Reticulum

fects the function of acidic vesicles (data not shown). Cells incubated at 20 C block protein traf®c beyond the Golgi apparatus (Fuller et al., 1985; Saraste et al., 1986). Pulse-radiolabeled class I molecules, chased at 20 C were also rescued from degradation (Figure 3C). Thus, the overexpressed class I molecules are not retained within the cell but are degraded in a post-Golgi compartment, presumably in the lysosomes. Traffic through the secretory pathway in overexpressor lines is normal Could class I overexpression cause a ``traf®c jam'' in the exocytic pathway? To address this question, the traf®c of Sindbis virus structural proteins, E1, pE2 and E2, were analyzed in NS0 (the parent cell line), Kb-high and Db-high cells. The conversion of pE2 to E2 occurs during transit of pE2 from the trans-Golgi/trans-Golgi network to the cell surface (Watson et al., 1991; Moehring et al., 1993) and served as an indicator of traf®c beyond the Golgi apparatus. The data revealed that pE2 synthesized during the pulse completely matures into E2 by four hours. The rate of this processing is almost identical when comparing the non-engineered NS0 and the engineered Kb-high and Db-high cell lines (Figure 3D). Further, the rate at which E1 appears in the culture supernatant, a measure for its arrival at the cell surface, is not affected by overexpression of class I molecules (Figure 3E). Thus, overexpression of class I molecules probably does not impair the traf®c of membrane proteins through the exocytic vesicles.

Conclusions The assembly and traf®c of MHC class I molecules in vivo has been delineated in great detail (reviewed by Joyce & Nathenson, 1996). However, the itinerary of these molecules after egress from the ER is not known. It is generally assumed that upon egress from the ER, class I molecules quickly reach the cell surface without requirement for positive sorting (Jackson et al., 1994). If this was true then all completely assembled class I molecules should accumulate at the cell surface. However, I ®nd that only a fraction (2 to 20%) of the overexpressed (up to 50-fold) completely assembled class I molecules are stably expressed on the plasma membrane. The regulated expression of class I molecules at the cell surface can be achieved by one of two mechanisms. One mechanism might involve rapid internalization of the majority of the overexpressed class I molecules that arrive at the cell surface soon after egress from the ER. This is followed by a slow return of class I back to the cell surface by recycling. Thus the two processes, internalization and recycling, would result in maintaining the majority of the completely assembled class I molecules inside the cell. An alternate mechanism might involve sorting of class I in the trans-Golgi or the trans-Golgi network. If sorting is accom-

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Traf®c Control Beyond the Endoplasmic Reticulum

plished by a saturable cytosolic factor(s), this would result in the controlled expression of class I molecules on the plasma membrane. The sorting of MHC class I molecules in the Golgi apparatus would represent a novel mechanism that could control the nature and quantity of cytosolic peptide antigens displayed at the cell surface. Such a control mechanism could in turn regulate CD8‡ T cell development and function.

Methods Generation of class I overexpressor lines in mammalian cells The full length cDNA of H-2Kb, Db and b2-ma were subcloned into the mammalian cell expression vector, pEE12. pEE12 encodes glutamine synthetase as the selection and ampli®cation marker as well as the human cytomegalovirus enhancer-promotor cassette as the transcription control elements. The transcription orientation of Kb, Db and b2-ma cDNA in the respective constructs pEE12.K, pEE12.D and pEE12.b2-m were analyzed by restriction mapping. A plasmid carrying cDNA encoding the heavy and light chains, as separate transcriptional units, was constructed by ligating a NheI-BglII fragment of pEE12.K with a BamHI-NheI fragment of pEE12.b2-m to obtain pEE12Kb or by ligating a NheI-BglII fragment of pEE12.b2-m with a BamHI-NheI fragment of pEE12.D to obtain pEE12Db. All enzymes and buffers were from New England Biolabs and Promega; DNA cloning protocols were as described (Sambrook et al., 1989). About 25 mg of NaeI linearized pEE12Kb or pEE12Db was electroporated into NS0 cells using a Gene Pulsar unit (BioRad). After ®ve minutes on ice, cells were resuspended in prewarmed (37 C) cell culture medium (DMEM) and plated at 104, 0.5  104 and 0.25  104 cells per well in microtiter plates. After 24 hours of recovery at 37 C, transfectants were selected in L-glutamine free DMEM containing 10% dialyzed FCS (foetal calf serum), sodium pyruvate, nucleosides, L-asparagine and L-glutamic acid (G-DMEM). Single auxotrophic colonies were harvested and expanded. H-2Kb and Db expression was monitored by cyto¯uorometry and immune precipitation. H-2Kb and Db gene ampli®cation was accomplished with 5, 10, 15, 20 and 25 mM methionine sulfoximine (Msx; Sigma Chemicals), an inhibitor of GS, in microtiter plates at 0.25  104 cells per well. Msx induces ampli®cation of the GS cDNA, thus co-amplifying 5100 kb of ¯anking DNA (Bebbington & Hentschel, 1987; Cockett et al., 1990). Cells from wells containing single isolated Msx resistant colonies were harvested and class I expression determined by ¯ow cytometry.

Flow cytometry Cell surface class I expression was determined using Y3 (Kb speci®c) and B22-249 (Db speci®c) mAb and analyzed with a FACScan ¯ow cytometer (Becton-Dickinson). The data were collected in a logarithmic mode and the geometric mean of ¯uorescence intensity was calculated after subtracting the background ¯uorescence obtained by staining with the anti-mouse immunoglobulin-FITC alone in the absence of speci®c mAb as described (Joyce et al., 1992).

Metabolic labelling, immunoprecipitation and analysis of glycoproteins Steady state labeling of cells and immune precipitations with Y3 and B22-249 were performed as described (Joyce et al., 1994a). Quantitation of the heavy and light chain bands was performed by densitometric scanning of the autoradiogram according to the manufacturer's instructions (Molecular Dynamics). L-Methionine or L-methionine and L-cysteine starved cells were pulsed with 250 mCi of [35S]methionine or [35S]methionine and [35S]cysteine for ten to 15 minutes. An aliquot representing 5  105 overexpressor cells or 5  106 non-engineered cells were quickly harvested into ice-cold phosphate-buffered saline (PBS) (0 minutes of chase). The remaining cells were chased in the presence of ten-fold excess of L-methionine or L-methionine and L-cysteine for the indicated times either at 37 C in the presence or absence of 50 mM ammonium chloride or at 20 C. To determine the glycosylation status of the protein, in Figure 3A, the immune complexes were eluted in 0.5 ml of 50 mM sodium citrate (pH 5.5) containing 0.1% SDS and 5 mU of endoglycosidase H (Boehringer Mannheim). After digestion for 18 hours at 37 C, the immune complexes were precipitated with 10% (v/v) TCA for four hours on ice, washed extensively with PBS and processed for SDS-PAGE. In Figure 3C, the immune precipitates were directly endoglycosidase H digested (1 to 2 mU in 20 ml of 50 mM sodium citrate (pH 5.5), 0.1% SDS buffer) for 18 hours at 37 C (Capps & Zuniga, 1993); the complexes were dissociated with an equal volume of twice concentrated SDS-PAGE sample buffer (Laemmli, 1970). To separate proteins by charge, the immune complexes were quickly rinsed in distilled water and dissociated with IEF sample loading buffer and resolved by vertical gel one dimensional IEF (Ploegh, 1995). Metabolic labeling, isolation and analysis of class I associated peptides Cells were metabolically labeled with [3H]tyrosine essentially as described (Sharma et al., 1996). H-2Kb and Db molecules were af®nity puri®ed over Y3 or B22-249 coupled protein A Sepharose columns; the associated self peptides were isolated by Centricon 3 ®ltration and fractionated by reversed-phase HPLC as described (Joyce et al., 1994a). Ten percent of the products of each puri®cation step were quantitated by scintillation counting (Beckman). The concentrated Centricon 3 ®ltrate was loaded onto a 1.0  250 mm C18 Nucleosil column (5 mm, 300  A; Alltech) in 0.1% (v/v) tri¯uroacetic acid (TFA) in distilled water using an automated HP 1090 HPLC unit (Hewlett Packard). Peptides were eluted by exchanging the loading buffer with 0.1% TFA in 90% acetonitrile over a period of 17 minutes. Analysis of Sindbis virus structural protein traffic Infection of NS0 and the overexpressor cells (2  107) was initiated by adsorption of virus to cells at a multiplicity of infection of 10 at 4 C for 60 minutes in DMEM containing 1% FCS. Cells were washed and resuspended in DMEM at 2.5  106 cells per ml and infection proceeded for eight to 12 hours at 37 C. Mock-infected (inoculum without virus) and infected cells were harvested and processed for metabolic labeling, chase and immunoprecipitation with heteroantiserum against Sindbis virus structural proteins, E1 or pE2 ‡ E2. Radiolabeled

1000 virus was isolated from the labeling supernatant at the end of each chase period by ultracentrifugation at 100,000 g for 60 minutes at 4 C. The virus pellet was extracted in 50 ml of 0.5% SDS for ®ve to ten minutes on ice and diluted to 0.5 ml with 20 to 50 mM Tris-Cl buffer (pH 8.0), containing 0.5% (w/v) Nonidet P-40, 150 mM NaCl, 1 mM EDTA and 20 mM phenylmethylsulfonyl ¯uroide. After clari®cation, the viral E1 glycoprotein was immunoprecipitated with a rabbit immune heteroantiserum. The structural proteins were separated by SDSPAGE and visualized by autoradiography. Affinity purification, peptide isolation and amino acid sequence analysis H-2Db molecules were af®nity puri®ed from detergent lysates of 5  109 Db-high cells and the associated self peptides were isolated and separated from the heavy and light chains by Centricon 3 ultra®ltration as described (Joyce et al., 1994a). Approximately 10% of the peptides in the Centricon 3 ®ltrate was subjected to pool sequencing by 12 automated cycles of Edman degradation using an ABI 477A sequencer (Applied Biosystems). Control peptide preparation included Centricon 3 ®ltrate of the material eluted from an anti-X8 column chromatography of Db-high detergent lysates. This preparation did not yield any signi®cant amounts of PTHamino acids upon Edman analysis.

Acknowledgements This work was initiated in Dr Stanley G. Nathenson's laboratory as a postdoctoral fellow supported by the Cancer Research Institute; Dr Stanley G. Nathenson's encouragement and support during this crucial period of my career are gratefully appreciated. I gratefully acknowledge M. H. Hunsinger for technical help; J. Bard for teaching me DNA cloning techniques; C. Bebbington and CellTech for providing pEE6 and pEE12 mammalian cell expression vectors; S. Kvist, G. Waneck and P. Kourilsky for Kb, Db and b2-ma cDNAs, respectively; M. D. Scharff for NS0 cells; K. Karre for RMA cells; S. G. Nathenson for class I, H-2Kb and Db speci®c antibodies; K. Hasenkrug for anti-IA speci®c mAb; M. J. Chorney for anti-b2-ma antiserum; C. M. Rice for Sindbis virus and Sindbis structural protein speci®c antisera; R. P. Machold for the vertical one dimensional IEF protocol; Y. Shi and R. H. Angeletti for sequencing the peptide pools; J. R. Bennink, H. L. Ploegh, D. Shields, P. Stanley, J. W. Yewdell and M. C. Zuniga for helpful discussions. This work is supported in parts by grants from the NIH (RO1 HL54977-01) and The Children's Miracle Network. S. J. is the recipient of the American Cancer Society's Junior Faculty Research Award.

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Edited by I. B. Holland (Received 1 July 1996; received in revised form 17 November 1996; accepted 22 November 1996)