The Journal of Cell Biology, Vol 111, 31-44, Copyright © 1990 by The Rockefeller University Press
Intracellular processing and transport of NH2-terminally truncated forms of a hemagglutinin-neuraminidase type II glycoprotein
MK Spriggs and PL Collins
Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Six amino-terminal deletion mutants of the NH2-terminally anchored (type II
orientation) hemagglutinin-neuraminidase (HN) protein of parainfluenza
virus type 3 were expressed in tissue culture by recombinant SV-40 viruses.
The mutations consisted of progressive deletions of the cytoplasmic domain
and, in some cases, of the hydrophobic signal/anchor. Three activities were
dissociated for the signal/anchor: membrane insertion, translocation, and
anchoring/transport. HN protein lacking the entire cytoplasmic tail was
inserted efficiently into the membrane of the endoplasmic reticulum but was
translocated inefficiently into the lumen. However, the small amounts that
were successfully translocated appeared to be processed subsequently in a
manner indistinguishable from that of parental HN. Thus, the cytoplasmic
domain was not required for maturation of this type II glycoprotein.
Progressive deletions into the membrane anchor restored efficient
translocation, indicating that the NH2-terminal 44 amino acids were fully
dispensable for membrane insertion and translocation and that a 10-amino
acid hydrophobic signal sequence was sufficient for both activities. These
latter HN molecules appeared to be folded authentically as assayed by
hemagglutination activity, reactivity with a conformation-specific
antiserum, correct formation of intramolecular disulfide bonds, and
homooligomerization. However, most (85-90%) of these molecules accumulated
in the ER. This showed that folding and oligomerization into a biologically
active form, which presumably represents a virion spike, occurs essentially
to completion within that compartment but is not sufficient for efficient
transport through the exocytotic pathway. Protein transport also appeared
to depend on the structure of the membrane anchor. These latter mutants
were not stably integrated in the membrane, and the small proportion
(10-15%) that was processed through the exocytotic pathway was secreted.
The maturation steps and some of the effects of mutations described here
for a type II glycoprotein resemble previous observations for prototypic
type I glycoproteins and are indicative of close similarities in these
processes for proteins of both membrane orientations.
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