The delta subunit of AP-3 is required for efficient transport of VSV-G from the trans-Golgi network to the cell surface

Abstract

Vesicular stomatitis virus glycoprotein (VSV-G) is a transmembrane protein that functions as the surface coat of enveloped viral particles. We report the surprising result that VSV-G uses the tyrosine-based di-acidic motif (-YTDIE-) found in its cytoplasmic tail to recruit adaptor protein complex 3 for export from the trans-Golgi network. The same sorting code is used to recruit coat complex II to direct efficient transport from the endoplasmic reticulum to the Golgi apparatus. These results demonstrate that a single sorting sequence can interact with sequential coat machineries to direct transport through the secretory pathway. We propose that use of this compact sorting domain reflects a need for both efficient endoplasmic reticulum export and concentration of VSV-G into specialized post-trans-Golgi network secretory-lysosome type transport containers to facilitate formation of viral coats at the cell surface.

Figure 1

Specific interaction of VSV-G and AP-3. (A) Two-hybrid analysis of the interaction of δ-adaptin (residues 578–825) with wild-type (YxDxE), AxDxE, and YxAxA sorting motifs found in the VSV-G cytoplasmic tail. β-Galactosidase activity of Y190 strains coexpressing the indicated VSV-G tail and δ-adaptin constructs was performed in triplicate. (B) Coimmunoprecipitation of VSV-G and APs. HeLa cells were transfected with either pAR wild-type VSV-G or the AxDxE or YxAxA mutants, and VSV-G was immunoprecipitated by using anti-VSV-G antibody. The VSV-G containing immunoprecipitates (Left) or 10% of total lysate used for immunoprecipitation (Right) were immunoblotted by using γ-, α-, and δ-adaptin antibodies. (C) Quantitation of recovery in B adjusted for total AP-3 in lysate.

Figure 2

AP-3 can facilitate TGN-PM transport of VSV-G. (A) The AP-3 fragment inhibits TGN to PM transport of VSV-G. Baby hamster kidney (BHK) cells were transfected with the pAR wild-type VSV-G and either the pCR3.1 (□) (mock) or pCR3.1-AP-3 fragment (AP-3*) (○). Cell surface (S) and intracellular forms are indicated (I). (Inset) AP-3* does not inhibit ER to Golgi transport of VSV-G. BHK cells were transfected with wild-type VSV-G and either pCR3.1 (mock) or the AP-3*, labeled, and processed for endo H resistance. (B) ER to Golgi transport of VSV-G at 20°C. Appearance of endo H-resistant forms of wild-type (YxDxE) (□) or the AxDxE (▵) and YxAxA (○) mutants in BHK cells was measured for the indicated time at 20°C. (C) TGN to PM transport of VSV-G. Appearance of biotinylated forms of wild-type (YxDxE) (□) or the AxDxE (▵) and YxAxA (○) mutants in BHK cells was measured for the indicated time at 37°C after either 100 min (YxDxE and AxDxE) or 240 min (YxAxA) at 20°C.

Figure 3

VSV-G but not HA uses AP-3 for its transport to cell surface. (A) Appearance of biotinylated forms of VSV-G in C57BL/6J (□) and Mocha (○) fibroblasts was measured at the indicated time at 37°C after a 100-min preincubation at 20°C. (Inset) Appearance of endo H-resistant forms of VSV-G in C57BL/6J and Mocha fibroblasts was measured after a 100-min preincubation at 20°C. (B) C57BL/6J (□) and Mocha (○) fibroblasts were transfected with HA, labeled for 20 min with [³⁵S]Met, chased with unlabeled Met for the indicated time at 37°C, digested with trypsin to cleave cell surface HA, and lysed as described (64). Total HA including uncleaved (HA0 representing intracellular HA) and cleaved (HA1 and HA2 representing cell surface HA) forms was immunoprecipitated with anti-HA antibody. The values for surface delivery of HA are expressed as a percent of HA1 + HA2/HA0 + HA1 + HA2.

Figure 4

VSV-G can compete with LAMP1 for AP-3-mediated packaging in the TGN. Uninfected (B and C) or infected HeLa cells (A and D) with VSV at 32°C as described (33) were incubated in the presence of mouse anti-LAMP1 mAb (C and D) for 45 min at 32°C, fixed, permeabilized, and processed for indirect immunofluorescence microscopy with the indicated antibodies as described (33). Images typical of >300 cells were examined for each indicated condition. Arrows in A indicate VSV-G in Golgi elements, arrowheads in B and D indicate distribution of LAMP1 containing punctate structures. Asterisked line indicates location of density profile (pixels) shown in C and D Insets. (Magnifications: ×63.)

Figure 5

VSV-G recruits AP-3 to the TGN. BHK cells were infected with the tsO45 VSV variant at 39.5°C for 4 h and subsequently transferred to ice (A and B) or incubated at 32°C for 1 (C and D), 5 (E and F) or 10 (G and H) min before transfer to ice. The distribution of VSV-G and AP-3 (δ-adaptin) were visualized by Texas red and Alexa 488, respectively. (Magnifications: ×63.)