Degradation of mutant influenza virus hemagglutinins is influenced by cytoplasmic sequences independent of internalization signals.
A mutant influenza virus hemagglutinin, HA+8, having a carboxyl-terminal extension of 8 amino acids that included 4 aromatic residues, was internalized within 2 min of arriving at the cell surface and was degraded quickly by a process that was inhibited by ammonium chloride. Through second-site mutagenesis, the internalization sequence of HA+8 was found to closely resemble the internalization signals of the transferrin receptor or large mannose 6-phosphate receptor. Comparison of the intracellular traffic of HA+8 and a series of other HA mutants that differed in their rates of internalization revealed a relation between the amount of the protein on the plasma membrane at steady state and the internalization rate that would be predicted if most of each protein recycled to the cell surface. However, there was no simple correlation between the internalization rate and the rate of degradation, indicating that transport to the compartment where degradation occurred was not simply a function of the concentration of the proteins in early endosomes. The internal populations of both HA+8, which was degraded with a t1/2 of 1.9 h, and HA-Y543, which was degraded with a t1/2 of 2.9 h, were found by cell fractionation and density-shift experiments to reside in early endosomes with little accumulation in lysosomes. A fluid-phase marker reached lysosomes 3-4-fold faster than these proteins were degraded. Degradation of these mutant HAs involved a rate-determining step in early endosomes that was sensitive to some feature of the protein that depended upon sequence differences in the cytoplasmic domain unrelated to the internalization signal.
Zwart
D E
DE
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas 72235-9038, USA.
Brewer
C B
CB
Lazarovits
J
J
Henis
Y I
YI
Roth
M G
MG
D000595:Amino Acid Sequence; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D006388:Hemagglutinins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D009975:Orthomyxoviridae / Q000502:physiology*
D006388:Hemagglutinins