The nature of topogenic sequences determines the transport competence of topological mutants of neutral endopeptidase-24.11.
Type II integral membrane proteins are anchored by a signal-peptide/membrane-anchor domain (SA domain) located near their N-terminus, whereas type I membrane proteins are anchored by stop-transfer sequences usually located near the C-terminus. In this study we have attempted to transform neutral endopeptidase-24.11 (EC 3.4.24.11; NEP), a type II membrane protein, into a type I membrane protein. Three type I mutant proteins were constructed by fusion of topogenic sequences to the C-terminus of SecNEP, a soluble form of NEP. The first two type I mutants, SecNEP-TMC and SecNEP-TMIC, were constructed by fusing in frame the cytosolic and SA domains of NEP to the C-terminus of SecNEP. These two fusion proteins differ only in the orientation of the cytosolic tail. The third type I mutant, SecNEP-ACE, was constructed by fusing in frame the stop-transfer and cytosolic domains of angiotensin I-converting enzyme (EC 3.4.15.1; ACE) to the C-terminus of SecNEP. Our results suggest that: (1) the NEP ectodomain can be anchored with a type I topology in the endoplasmic reticulum (ER) membrane by both NEP and ACE topogenic sequences; (2) SecNEP-TMC and SecNEP-TMIC were transport-incompetent and needed proteolytic cleavage in the C-terminal region to leave the ER, whereas SecNEP-ACE was transported out of the ER as a type I membrane protein. Therefore we concluded that the nature of topogenic sequences determines the transport-competence of topological mutants of neutral endopeptidase-24.11.
D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000378:metabolism; D015151:Immunoblotting; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D015260:Neprilysin / Q000737:chemistry* / Q000378:metabolism*; D007703:Peptidyl-Dipeptidase A / Q000235:genetics; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection; D001619:beta-N-Acetylhexosaminidases / Q000378:metabolism
D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D006026:Glycoside Hydrolases; D001619:beta-N-Acetylhexosaminidases; D007703:Peptidyl-Dipeptidase A; D015260:Neprilysin