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J Biol Chem, Vol. 274, Issue 10, 6776-6782, March 5, 1999
From the Department of Molecular and Cell Biology, The University
of Connecticut, Storrs, Connecticut 06269
Many integral membrane proteins contain an
amino-terminal segment, often referred to as an N-tail, that is
translocated across a membrane. In many cases, translocation of the
N-tail is initiated by a cleavable, amino-terminal signal peptide. For
N-tail proteins lacking a signal peptide, translocation is initiated by
a transmembrane segment that is carboxyl to the translocated segment.
The mechanism of membrane translocation of these segments, although
poorly understood, has been reported to be independent of the protein
secretion machinery. In contrast, here we describe alkaline phosphatase
mutants containing artificial transmembrane segments that demonstrate
that translocation of a long N-tail across the membrane is dependent
upon SecA, SecB, and the electrochemical potential in the absence of a
signal peptide. The corresponding mutants containing signal peptides
also use the secretion machinery but are less sensitive to inhibition
of its components. We present evidence that inhibition of SecA by sodium azide is incomplete even at high concentrations of inhibitor, which suggests why SecA-dependent translocation may not
have been detected in other systems. Furthermore, by varying the charge around the transmembrane segment, we find that in the absence of a
signal peptide, the orientation of the membrane-bound alkaline phosphatase is dictated by the positive inside rule. However, the
presence of a signal peptide is an overriding factor in membrane orientation and renders all mutants in an
Nout-Cin orientation.
An Artificial Transmembrane Segment Directs SecA, SecB, and
Electrochemical Potential-dependent Translocation of a Long
Amino-terminal Tail
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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