Fig. 5. Changes in any of the four TM domains prevent UPIb from exiting from the ER. (A) Non-permeabilized 293T cells expressing UPIb, UPIa or the UPIa/UPIb chimeras indicated were analyzed by immunofluorescence microscopy. The chimeras Ib/a2, Ib/a4 and Ib/a8 were not transported to the plasma membrane, but retained in the ER (not shown). (B) Point mutants of UPIb, where the Glu32 or Glu102 were replaced by Ala (IbE32A, IbE102A), or Gln (UPIbE32Q, Ib102EQ), respectively, and Tyr99 by Ala (IbY99A), were expressed in 293T cells, followed by immunostaining of non-permeabilized cells. IbE102A was not able to reach the cell surface, indicating in addition to TM1, TM2 and TM4, the glutamic acid residue in TM3 is important for the proper assembly of the TM helices of UPIb.