Endocytosis of chimeric influenza virus hemagglutinin proteins that lack a cytoplasmic recognition feature for coated pits.

Abstract

Abstract. The influenza virus A/Japan/305/57 hemagglutinin (HA) can be converted from a protein that is essentially excluded from coated pits into one that is internalized at approximately the rate of uptake of bulk membrane by replacing the HA transmembrane and cytoplasmic sequences with those of either of two other glycoproteins (Roth et al., 1986. J. Cell Biol. 102:1271-1283. Toddeutify more precisely the foreign amino acid sequences responsible for this change in HA traffic, DNA sequences encoding the transmembrane (TM) or cytoplasmic (CD) domains of either the G glycoprotein of vesicular stomatitis virus (VSV) or the gC glycoprotein of herpes simplex virus were exchanged for those encoding the analogous regions of wild type HA (HA wt). HA-HA-G and HA-HA-gC, chimeras that contain only a foreign CD, resembled HA wt in having a long residence on the cell surface and were internalized very slowly. HA-HA-gC was indistinguishable from HA in our assays, whereas twice as much HA-HA-G was internalized as was HA wt. However, HA-G-HA, containing only a foreign TM, was internalized as efficiently as was HA-G-G, a chimeric protein with transmembrane and cytoplasmic sequences of VSV G protein. Conditions that blocked internalization through coated pits also inhibited endocytosis of the chimeric proteins. Although the external domains of the chimeras were less well folded than that of the wild type HA, denaturation of the wild type HA external domain by treatment with low pH did not increase the interaction of HA with coated pits. However, mutation of four amino acids in the TM of HA allowed the protein to be internalized, indicating that the property that allows HA to escape endocytosis resides in its TM. These results indicate that possession of a cytoplasmic recognition feature is not required for the internalization of all cell surface proteins and suggest that multiple mechanisms for internalization exist that operate at distinctly different rates.Part of the process of communicating with their external environment, cells maintain an active traffic of membranes moving between the cell surface and internal organelles. Solutes are absorbed through fluid-phase endocytosis, nutrients and hormones are concentrated and internalized by receptor-mediated endocytosis, and resident proteins of the plasma membrane are removed for degradation in lysosomes. Cellular pathogens, like many viruses, use these endocytic processes to enter the cell. It is clear that there are at least several distinct pathways for this traffic (Watts and Marsh, 1992), but the exact number of pathways and the relations between them are not currently known. However, in cells that are not undergoing major changes in shape, such as those that occur during locomotion or phagocytosis, a major pathway

The Rous Sarcoma Virus Env Glycoprotein Contains a Highly Conserved Motif Homologous to Tyrosine-Based Endocytosis Signals and Displays an Unusual Internalization Phenotype

“…Unexpectedly, the endocytosis rate exhibited by Env-S19/Y190A (0.3%/min) is significantly lower (P Յ 0.005) than that of the wild type. This very slow rate is similar to that of influenza virus HA (0.2%/min in CV-1 cells [32]), a protein shown to be actively excluded from clathrin-coated pits (6,33,55). To our knowledge this is the first study in which substitution of a tyrosine within a known YXXØ endocytosis motif resulted in a decrease in the uptake rate to below that of bulk uptake.…”

“…Since the rates of bulk membrane and protein uptake in CV-1 cells and other cell types have been described to be 1 to 2% per min (32,47), it was somewhat surprising that RSV Env, which contains a YXXØ endocytosis motif consensus sequence, showed an uptake rate equal to or slightly lower than that reported for the bulk membrane uptake. Internalization of proteins at the rate of membrane turnover does not require any specific uptake signals since no active concentration into coated pits occurs (for a review, see reference 63).…”

“…lope glycoprotein with a wild-type TM is indeed endocytosed from the surfaces of CV-1 cells, but at a slow rate (1.1%/min) reminiscent of that of membrane bulk uptake (22,32,47) despite the presence of the YRKM motif. In contrast, a quantitative analysis of previously described MSD mutants Env-26 (L165R) and Env-palm#7 (C167G) that exhibit significantly reduced steady-state surface expression (27) revealed a much more rapid rate of endocytosis, with an average of 74 and 47%, respectively, being internalized within the initial 10 min of a chase.…”

“…Thus, for HA, there might exist some correlation between protein sequestering into certain plasma membrane domains and exclusion from clathrin vesicles. In this case, however, functional YXXØ motifs introduced into the cytoplasmic domain of HA appear to act dominantly over the MSD retention signals since rapid endocytosis now takes place (30,32,33,35). We, therefore, examined whether the different RSV Env MSD and C-terminal tail mutants might display a differential pattern of DIG association (i.e., strong association for Env-S19-Y190A, low or no association for Env-S19-26 and Env-S19-palm#7, respectively), which could control the extent to which the proteins are excluded from clathrin-coated pits, particularly since Melkonian et al (40) had shown that the extent of palmitoylation can modulate association with DIGs.…”

“…In contrast, wild-type HA, which internalizes at a rate of approximately 0.2%/min in CV-1 cells (32), was found to be actively excluded from coated pits (6,33,55) by virtue of sequences within its MSD (32). Mutants with certain changes in that part of the MSD in contact with the outer lipid leaflet were no longer excluded (32). Some, but not all, of these HA MSD mutants also showed a loss of HA's efficient association with FIG.…”