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. 2017 Jul 28;6:e26067. doi: 10.7554/eLife.26067

Figure 2. Downstream residues of gp160 regulate signal-peptide cleavage.

(A) Schematic of different GFP reporter constructs generated for imaging and immunoblot experiments. All reporters have the identical monomeric GFP-KDEL cassette. For ER GFP, the GFP-KDEL cassette is preceded by the bovine prolactin signal peptide (open box SP). The reporters with the HIV Env signal peptide (grey box SP) are fused to one or more amino acids of the mature gp120 domain followed by the GFP-KDEL cassette. (B) Western Blot analysis of control constructs (GFP-KDEL, ER-GFP) or gp160 signal peptide with 1-30 downstream residues (SP +1, …, 30). GFP-KDEL and SP +1 run lower on gel as they lack the additional residues downstream of the cleavage site. Ru: unprocessed molecules with signal peptide still attached; Rc: signal peptide-cleaved molecules. All images shown are representative of at least two independent experiments performed with fresh cells and transfections (biological replicates).

Figure 2.

Figure 2—figure supplement 1. Characterization of SP-GFP fusion constructs.

Figure 2—figure supplement 1.

(A) Sequence alignment of GFP-fusion constructs. Protein-sequence alignment of GFP-gp120 fusion constructs used in Figure 2. Only the first 9 residues of GFP are included for brevity. The arrow indicates the predicted signal-peptide cleavage site. All constructs contain a C-terminal ER-retrieval signal KDEL. (B) Fluorescence microscopy of Cos-7 cells expressing GFP constructs containing ER-retention signal KDEL. GFP-KDEL: cytosolic control GFP; ER-GFP: ER-resident GFP with the signal peptide of hen-egg lysozyme; SP +1: GFP with gp160 signal peptide and one gp160 residue as linker; SP +10: GFP with gp160 signal peptide and ten gp160 residues; ER-RFP: ER marker. Scale bar = 10 μm. (C) ER-GFP, SP +1 and SP +10 were analyzed by pulse chase as in Figure 1A. (D) Quantifications from C. *: background band.