Phosphatidylserine-binding protein lactadherin inhibits protein translocation across the ER membrane

•Lactadherin inhibited the cell-free protein translocation across the ER membrane.•Lactadherin suppressed membrane targeting, translocation initiation, and movement.•Binding deficient mutant of lactadherin showed no effects.•Lactadherin did not enhance movement of positive charges on protein. Secretory and membrane proteins are translocated across and inserted into the endoplasmic reticulum membrane via translocon channels. To investigate the effect of the negatively-charged phospholipid phosphatidylserine on the translocation of nascent polypeptide chains through the translocon, we used the phosphatidylserine-binding protein lactadherin C2-domain. Lactadherin inhibited targeting of nascent chain to the translocon by signal sequence and the initiation of translocation. Moreover, lactadherin inhibited the movement of the translocating polypeptide chain regardless of the presence or absence of positively-charged residues. Phosphatidylserine might be critically involved in translocon function, but it is not a major determinant for translocation arrest of positively-charged residues.