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Fig. 3. Amino acids of the C-terminal domain of UPIb, including a crucial tyrosine residue, are tested for their function as an ER exit signal. (A) 293T cells expressing UPIb/a9, a chimeric protein in which the cytoplasmic C-terminal domain was replaced with that of UPIa. No immunostaining was observed in intact cells. (B) An alanine scan was performed on the C-terminal domain of UPIb, generating the Ala mutants UPIbA1-10. Similarly, Tyr253 was replaced with phenylalanine (UPIbF7). (C) Non-permeabilized 293T cells expressing the mutant proteins listed in B were immunostained. (D) Deletion mutants of UPIb were constructed by removing step-wise seven amino acids of the C-terminal domain 9. (E) 293T cells were transfected with UPIb or the deletion mutants indicated in D, followed by immunostaining of the non-permeabilized cells with a polyclonal anti-UPIb antibody. Only the deletion mutants that retain at least four amino acids of the C-terminal domain exit from the ER and are expressed at the cell surface. Deletion mutants Ib4 to Ib7 were retained in the ER as could be seen by immunostaining of permeabilized cells (data not shown).

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