The E3/19K protein of adenovirus type 2 is a resident of the ER. Immediately after synthesis it binds to human major histocompatibility complex class I antigens and prevents their departure from the ER compartment. The ER retention signal of the E3/19K protein is contained within the 15 amino acids that protrude on the cytoplasmic side at the carboxy terminus of the protein. To define the ER retention sequence in more detail, we have generated 10 mutants of the E3/19K protein that differ only within this segment. Analysis of the rate of intracellular transport and cell surface expression of HLA antigens associated to these mutants, show that the sequences Ser-Phe-Ile, located in the middle of the 15-residue segment and Met-Pro, at the extreme carboxy terminus, are crucial for retention. Four charged residues, Asp-Glu-Lys-Lys, are located between these two retention elements but are of little or no importance. The basic cluster of amino acids close to the membrane also has some effect on retention. Thus, the retention signal of the E3/19K protein is not a contiguous sequence of amino acids but has a complex spatial arrangement.

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