The Journal of Cell Biology, Vol 104, 1705-1714, Copyright © 1987 by The Rockefeller University Press
The influenza hemagglutinin insertion signal is not cleaved and does not halt translocation when presented to the endoplasmic reticulum membrane as part of a translocating polypeptide
J Finidori, L Rizzolo, A Gonzalez, G Kreibich, M Adesnik and DD Sabatini
The co-translational insertion of polypeptides into endoplasmic reticulum
membranes may be initiated by cleavable amino-terminal insertion signals,
as well as by permanent insertion signals located at the amino-terminus or
in the interior of a polypeptide. To determine whether the location of an
insertion signal within a polypeptide affects its function, possibly by
affecting its capacity to achieve a loop disposition during its insertion
into the membrane, we have investigated the functional properties of
relocated insertion signals within chimeric polypeptides. An artificial
gene encoding a polypeptide (THA-HA), consisting of the luminal domain of
the influenza hemagglutinin preceded by its amino-terminal signal sequence
and linked at its carboxy-terminus to an intact prehemagglutinin
polypeptide, was constructed and expressed in in vitro translation systems
containing microsomal membranes. As expected, the amino-terminal signal
initiated co-translational insertion of the hybrid polypeptide into the
membranes. The second, identical, interiorized signal, however, was not
recognized by the signal peptidase and was translocated across the
membrane. The failure of the interiorized signal to be cleaved may be
attributed to the fact that it enters the membrane as part of a
translocating polypeptide and therefore cannot achieve the loop
configuration that is thought to be adopted by signals that initiate
insertion. The finding that the interiorized signal did not halt
translocation of downstream sequences, even though it contains a
hydrophobic region and must enter the membrane in the same configuration as
natural stop-transfer signals, indicates that the HA insertion signal lacks
essential elements of halt transfer signals that makes the latter effective
membrane-anchoring domains. When the amino- terminal insertion signal of
the THA-HA chimera was deleted, the interior signal was incapable of
mediating insertion, probably because of steric hindrance by the folded
preceding portions of the chimera. Several chimeras were constructed in
which the interiorized signal was preceded by polypeptide segments of
various lengths. A signal preceded by a segment of 111 amino acids was also
incapable of initiating insertion, but insertion took place normally when
the segment preceding the signal was only 11-amino acids long.(ABSTRACT
TRUNCATED AT 400 WORDS)
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