Major histocompatibility complex (MHC)-encoded HAM2 is necessary for antigenic peptide loading onto class I MHC molecules.

The mutant murine lymphoma cell line RMA-S is unable to present endogenous antigens due to its inability to efficiently assemble class I major histocompatibility complex molecules and antigenic peptides. Therefore, it has been suggested that RMA-S cells are defective either in peptide generation or in peptide transport into the endoplasmic reticulum, where class I major histocompatibility complex molecule assembly is believed to occur. As proteasomes and the putative peptide transporters HAM1 and HAM2 have been implicated in class I antigen processing, we have investigated their expression in RMA-S and its wild-type counterpart RMA. Both proteasomes and HAM1 proteins are expressed at similar levels and show identical subcellular distributions in the two cell lines. However, only one copy of the HAM2 gene is present in RMA-S cells, and it contains a point mutation that leads to a premature stop codon. Thus, the HAM2 protein is absent from RMA-S cells. These data demonstrate that HAM2 is essential for peptide loading onto class I molecules.

The mutant murine lymphoma cell line RMA-S is unable to present endogenous antigens due to its inability to efficiently assemble class I major histocompatibility complex molecules and antigenic peptides. Therefore, it has been suggested that RMA-S cells are defective either in peptide generation or in peptide transport into the endoplasmic reticulum, where class I major histocompatibility complex molecule assembly is believed to occur. As proteasomes and the putative peptide transporters HAMl and HAM2 have been implicated in class I antigen processing, we have investigated their expression in RMA-S and its wild-type counterpart RMA. Both proteasomes and HAMl proteins are expressed at similar levels and show identical subcellular distributions in the two cell lines. However, only one copy of the HAM2 gene is present in RMA-S cells, and it contains a point mutation that leads to a premature stop codon. Thus, the HAM2 protein is absent from RMA-S cells. These data demonstrate that HAM2 is essential for peptide loading onto class I molecules.
Although the structure and expression of the genes encoding P2-microglobulin and MHC' class I heavy chains are normal in RMA-S cells (I),' low cell surface expression of class I molecules has been observed (2, 3). The addition of synthetic, antigenic class I-binding peptides to RMA-S cells has restored surface expression of class I molecules and induced assembly of RMA-S-derived class I molecules in vitro * This work was supported by the National Institutes of Health.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. (3, 4). Therefore, the deficient assembly and intracellular transport of class I molecules in RMA-S cells might have been due to lack of class I-binding peptides resulting from defective antigen processing. Recent data have demonstrated that proteasomes (5), which represent the major extralysosomal proteolytic system (6), contain two MHC-encoded subunits (7-11), rendering the proteasome a prime candidate for generating antigenic peptides for class I molecules. Two genes called HAMl and HAM2 (12) in the mouse, which are members of the traffic ATPases superfamily of transporters (13), have been mapped in close vicinity to the MHC-encoded proteasomal subunits (12, 14). The rat homologues and the human homologues of HAMl and HAM2 are named MTPl and MTPZ (15) and PSFl and PSF2 (16), respectively. Their involvement in the transport of peptides across the ER membrane has been discussed (12, 14-20). In the present study, we have investigated the expression of proteasomal subunits and the putative peptide transporters HAMl and HAM2 in RMA-S and its wild-type counterpart RMA.

RESULTS AND DISCUSSION
T o examine if proteasomes are different in RMA and RMA-S cells, we immunoprecipitated metabolically labeled proteasomes from the two cell lines and separated the subunits by two-dimensional gel electrophoresis. The polypeptide patterns were identical, including the levels, charges, and molecular weights of the two MHC-encoded subunits (data not shown), which strongly suggested that proteasomes from RMA and RMA-S do not differ. If peptide generation is normal in RMA-S cells, it seemed likely that peptide transport from the cytoplasm into the ER is defective in RMA-S cells. The human cell line LCL 721.134, with a phenotype similar to that of RMA-S, had previously been shown to revert to the wild-type phenotype following transfection with PSFl (20), the human homologue of HAMl. Therefore, we investigated the expression of HAMl in RMA and RMA-S cells by immunoprecipitation and immunofluorescence staining using two antisera raised against a peptide corresponding to the carboxyl-terminal region of HAMl and a purified recombinant protein containing the entire putative ATP-binding domain of HAM1, respectively. Both antisera specifically recognized an interferon y-inducible 75-kDa protein as revealed by immunoprecipitation and sodium dodecyl sulfatepolyacrylamide gel electrophoresis. Immunofluorescence staining occurred in the perinuclear area representing the ER (data not shown). However, no difference was found with respect to size, charge, level of expression, and immunofluorescence staining pattern of HAMl between RMA and RMA-S cells. Furthermore, it has been demonstrated that the HAMl mRNA is expressed at similar levels in the two cell lines (14), and sequence analysis of the HAMl cDNA did not reveal any difference (data not shown). This indicates that the sequence and expression level of HAMl is identical in RMA and RMA-S cells, which may explain the findings that neither transfection of RMA-S with the rat homologue MTPl (15) nor transfection with the HAMl-containing cosmid 5.10 (12, 21) restored the surface expression of class I molecules (22).2 Consequently, the RMA-S phenotype must be caused by mutations in another gene.
It has also been reported that the MHC region contains a   Loading onto Class I Molecules methanesulfonate (23, 24). Therefore, we isolated and completely sequenced cDNA clones corresponding to HAM2 from RMA and RMA-S cDNA libraries. The sequence of HAM2 from RMA cells, shown in Fig. 1, contains an open reading frame of 702 residues, which encompasses the two short amino acid sequence stretches (positions 495-566 and 605-637) previously published (12). Fig. 2 shows that the HAM2 sequence displays 91% identity with the rat MTP2 sequence (22) and 77% identity with the human PSF2 sequence (25), while the identity with the HAMl sequence (12)* is less pronounced (39%). The HAM2 sequence, like the HAMl sequence, also displays striking homologies to other members of the traffic ATPases superfamily of transporters (not shown) and contains the typical a-@-a nucleotide binding motifs (26, 27) (amino acid residues 502-510 and 618-630 in Fig. 1). The sequence analysis of the HAM2 cDNA from RMA-S revealed only one difference compared with the RMA sequence at nucleotide position 97, which displayed a C to T transition (Fig. 3). This mutation introduces a premature stop codon. Several independent cDNA clones contained the same mutation at position 97 and no clone displayed the RMA sequence. Furthermore, the same mutation was found by directly sequencing PCR products obtained from reverse transcribed mRNA of RMA-S cells. No evidence of a mixed mRNA population consisting of both normal and mutated sequences was found (e.g. in Fig. 3). To investigate whether the RMA-S cells were homo-or hemizygous for the mutation, we amplified a region of the HAM2 gene corresponding to nucleotides 3-151 of HAM2 from RMA and RMA-S genomic DNA. The amplified DNA was separated by electrophoresis with or without prior digestion with AurII. This enzyme should cleave the mutated but not the wild-type HAM2 DNA fragment as the mutation created the recognition sequence CCTAGG for the enzyme AurII. Fig. 4 demonstrates that the HAM2 genomic DNA fragment derived from RMA-S was completely cleaved, while the fragment from RMA remained intact following the enzymatic treatment. The exclusive presence of a point mutation (C to T) in the HAM2 gene of RMA-S was further confirmed by directly sequencing the PCRamplified HAM2 genomic DNA and by Southern hybridization analysis of AurII-digested genomic DNA prepared from RMA and RMA-S (data not shown).

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Because it is unlikely that RMA-S cells are homozygous for the same point mutation in the HAM2 gene, this strongly suggests that RMA-S cells contain only one copy of the HAM2 gene and that the HAM2 protein is absent in RMA-S cells. On the contrary, Powis et al. (22) showed that a 65-kDa protein, which they identified as the HAM2 protein on the basis of its reactivity with an aMTP2 peptide (the NHpterminal MTPB sequence MALSHJRPWA(LC)) antiserum, was present in both RMA-S and RMA cells. However, a comparison of the NHp-terminal sequences of MTP2 and HAM2 reveals differences in 3 out of 10 amino acid residues (underlined; see Fig. 2). Furthermore, the HAM2 protein has a predicted molecular mass of 77.4 kDa, calculated from the translated cDNA sequence of HAM2. Therefore, this 65-kDa protein present in RMA-S is unlikely to be the HAM2 protein. The present data are consistent with the view that the absence of the HAM2 protein in RMA-S cells generates the defect in the assembly and intracellular transport of class I molecules. This would implicate HAM2 as an essential component of the peptide-loading machinery for class I molecules.  1 and H contain molecular weight standard (I-kh ladder, RRL). Fragment sizes are indicated in base pairs on the right. DNA fragments were electrophoretically separated on a 6% polyacrylamide gel using Tris-acetate buffer. Template genomic DNA for PCR was prepared from HMA and RMA-S using genomic DNA isolation kit (BRL). precipitated with PSFl by using a PSF1-specific antiserum (28). As HAMl apparently has a normal half-life and subcellular distribution in RMA-S cells, the two proteins could fold independently of each other. Thus, the possible interaction between HAMl and HAM2 may be a regulated event, which may activate the function of the putative peptide transporter.

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are grateful to Dr. .I. c . Howard for communicating the MTI'2 sequence prior to puhlication and Drs. M. .Jackson and I,. Karlsson for valuahle discussions and unpublished data.