Targeting of a short peptide derived from the cytoplasmic tail of the G1 membrane glycoprotein of Uukuniemi virus (Bunyaviridae) to the Golgi complex - PubMed (original) (raw)
Targeting of a short peptide derived from the cytoplasmic tail of the G1 membrane glycoprotein of Uukuniemi virus (Bunyaviridae) to the Golgi complex
A M Andersson et al. J Virol. 1998 Dec.
Abstract
Members of the Bunyaviridae family acquire an envelope by budding through the lipid bilayer of the Golgi complex. The budding compartment is thought to be determined by the accumulation of the two heterodimeric membrane glycoproteins G1 and G2 in the Golgi. We recently mapped the retention signal for Golgi localization in one Bunyaviridae member (Uukuniemi virus) to the cytoplasmic tail of G1. We now show that a myc-tagged 81-residue G1 tail peptide expressed in BHK21 cells is efficiently targeted to the Golgi complex and retained there during a 3-h chase. Green-fluorescence protein tagged at either end with this peptide or with a C-terminally truncated 60-residue G1 tail peptide was also efficiently targeted to the Golgi. The 81-residue peptide colocalized with mannosidase II (a medial Golgi marker) and partially with p58 (an intermediate compartment marker) and TGN38 (a trans-Golgi marker). In addition, the 81-residue tail peptide induced the formation of brefeldin A-resistant vacuoles that did not costain with markers for other membrane compartments. Removal of the first 10 N-terminal residues had no effect on the Golgi localization but abolished the vacuolar staining. The shortest peptide still able to become targeted to the Golgi encompassed residues 10 to 40. Subcellular fractionation showed that the 81-residue tail peptide was associated with microsomal membranes. Removal of the two palmitylation sites from the tail peptide did not affect Golgi localization and had only a minor effect on the association with microsomal membranes. Taken together, the results provide strong evidence that Golgi retention of the heterodimeric G1-G2 spike protein complex of Uukuniemi virus is mediated by a short region in the cytoplasmic tail of the G1 glycoprotein.
Figures
FIG. 1
Schematic representation of the different cDNA constructs used. (A) CD4-C81 is a chimeric protein in which the cytoplasmic tail of CD4 has been replaced by the cytoplasmic tail of G1 lacking the 17-residue internal signal sequence (ss) of G2. G1-tail and G1-tail-myc represent the cytoplasmic tail of G1 expressed as an 81-residue peptide, with or without a 10-residue c-myc tag. The G1 tail-myc was progressively deleted from the N terminus by 10 residues at a time, whereas the peptide from residues 10 to 81 was progressively deleted from the C terminus likewise by 10 residues at a time. (B) Residues 1 to 81 of the G1 tail were fused to either the C or N terminus of GFP, while residues 1 to 60 were fused only to the N terminus. When they were fused to the C terminus, a c-myc epitope tag was added to the C-terminal end. (C) Amino acid sequence of the 81-residue cytoplasmic tail of G1. The two cysteines at positions 25 and 28, known to become palmitylated in the intact G1 protein, are in bold and underlined.
FIG. 2
Colocalization of G1 and CD4-C81 with mannosidase II by immunofluorescence microscopy. G1 and CD4-C81 were expressed in BHK21 cells by using the SFV system. At 6 h posttransfection, the cells were treated for 3 h with CHX and then either permeabilized with Triton X-100 (a, b, d, and e), or left untreated for detection of surface staining (c and f). The cells were indirectly stained with a monoclonal antibody against G1 (a and c) or a monoclonal antibody against CD4 (d and f). Panels b and e show the cells in panels a and d double stained with a polyclonal antiserum against the Golgi marker protein mannosidase II (man II).
FIG. 3
Intracellular localization of GFP and GFP-G1 tail chimeras by immunofluorescence microscopy. GFP and the three GFP-G1 tail chimeras shown in Fig. 1B were expressed in BHK21 cells by using the SFV system. At 6 h posttransfection, the cells were treated for 4 h with CHX and permeabilized with Triton X-100. GFP was visualized by virtue of its autofluorescence (a, b, d, and f). The same cells were indirectly stained with a monoclonal antibody against the Golgi marker CTR-433 (c, e, and g).
FIG. 4
Colocalization of G1 tail-myc with marker proteins by confocal laser-scanning immunofluorescence microscopy. The myc-tagged G1 tail peptide was expressed in BHK21 cells by using the SFV system. (A, B, D, E, G, and H) At 6 h posttransfection, the cells were treated for 3 h with CHX before being double stained with a monoclonal antibody against the myc tag (A, D, and G) or with a polyclonal antiserum against the Golgi marker mannosidase II (man II), (B) against the ERGIC marker p58 (E), or against the SFV nonstructural protein nsP3, known to induce and associate with type I cytopathic vacuoles. (C, F, and I) Merged images of the double-stained cell.
FIG. 5
Colocalization of G1 tail-myc with marker proteins by immunofluorescence microscopy. The myc-tagged G1 tail peptide was expressed in BHK21 (A and B), NRK (C and D), or HeLa (E to H) cells by using the SFV system. At 6 h posttransfection, the cells were treated for 3 h with CHX before being double stained with a monoclonal antibody against the myc-tag (A, C, E, and G) or with a polyclonal antiserum against the ER marker calnexin (CN) (B), against the TGN marker TGN38 (D), or against the endosomal marker transferrin receptor (TfR) (F) or the lysosomal marker lamp-1 (H).
FIG. 6
Effect of BFA on the distribution of the G1 tail-myc peptide. G1 tail-myc was expressed in BHK21 cells by using the SFV system. At 6 h posttransfection, the cells were treated for 2 h with CHX (A and B) before being subjected to BFA treatment for 1 h in the presence of CHX (C and D). The BFA was washed out for 1 h in the presence of CHX (E and F). Permeabilized cells were double stained with a monoclonal antibody (Mab) against the myc tag (A, C, and E) and a polyclonal antiserum against mannosidase II (man II Pab) (B, D, and F).
FIG. 7
Analysis of the G1 tail-myc and the N- and C-terminally truncated G1 tail peptides by SDS-PAGE. The peptide constructs described in Fig. 1A were expressed in BHK21 cells. At 5.5 h posttransfection, the cells were starved in methionine-free medium for 45 min and then labeled with 0.14 mCi of [35S]pro-mix per ml for 20 min (lanes 1 to 7). At 3 h posttransfection, the G1 tail-myc was labeled in parallel for 5 h with 0.5 mCi of [9,10(n)-3H]palmitic acid per ml (lane 8). The cell lysates were immunoprecipitated with a monoclonal antibody against the myc tag and analyzed by SDS-PAGE (15% polyacrylamide) followed by fluorography. The positions of molecular weight (mw) markers are shown to the left in thousands.
FIG. 8
Intracellular localization of G1 tail-myc and mutated and truncated G1 tail peptides by immunofluorescence microscopy. In panel C (C25,28A), the two cysteines at position 25 and 28 were mutated to alanines to prevent palmitylation. The G1 tail constructs were expressed in BHK21 cells by using the SFV system. At 6 h posttransfection, the cells were treated for 3 h with CHX before being double stained with a monoclonal antibody against the myc tag (A, C, E, G, I, and K) or a polyclonal antiserum against mannosidase II (man II) (B, D, F, H, J, and L).
FIG. 9
Association of G1 tail-myc and G1 tail-myc C25,28A with membranes. The G1 tail-myc (A) and G1 tail-myc C25,28A (B) peptides were expressed in BHK21 cells by using the SFV system. Mock-transfected cells served as controls. At 5.5 h posttransfection, the cells were starved in methionine-free medium for 45 min and labeled with [35S]pro-mix for 20 min. Immediately after the labeling (lanes 1, 2, and 4 through 6) or following a chase for 1 h (lanes 7 and 8), the cells were homogenized and subjected to subcellular fractionation as described in Materials and Methods. The cell lysate (L), postnuclear supernatant (PNS), membranes (P), and supernatant (S) were immunoprecipitated with a monoclonal antibody against the myc tag, and the precipitates were analyzed by SDS-PAGE (15% polyacrylamide) followed by fluorography. Lanes 1 and 2 show mock-transfected cells used as controls. Lane 3 shows the total amount of expressed peptides in the cell lysate.
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