A transmembrane domain (TMD) at the N-terminus of a membrane protein is a signal 
sequence that targets the protein to the endoplasmic reticulum (ER) membrane. 
Proline is found more frequently in TM helices compared to water-soluble 
helices. To investigate the effects of proline on protein translocation and 
integration in mammalian cells, we made proline substitutions throughout the TMD 
of dipeptidyl peptidase IV, a type II membrane protease with a single TMD at its 
N-terminus. The proteins were expressed and their capacities for targeting and 
integrating into the membrane were measured in both mammalian cells and in vitro 
translation systems. Three proline substitutions in the central region of the 
TMD resulted in various defects in membrane targeting and/or integration. The 
replacement of proline with other amino acids of similar hydrophobicity rescued 
both the translocation and anchoring defects of all three proline mutants, 
indicating that conformational change caused by proline is a determining factor. 
Increasing hydrophobicity of the TMD by replacing other residues with more 
hydrophobic residues also effectively reversed the translocation and integration 
defects. Intriguingly, increasing hydrophobicity at the C-terminal end of the 
TMD rescued much more effectively than it did at the N-terminal end. Thus, the 
effect of proline on translocation and integration of the TMD is not determined 
solely by its conformation and hydrophobicity, but also by the location of 
proline in the TMD, the location of highly hydrophobic residues, and the 
relative position of the proline to other proline residues in the TMD.