TGN budding of ISGs is not affected by VHS-GAT-GFP and GGA1-GFP. PC12 cells were transfected with either VHS-GAT-GFP or GGA1-GFP and 24 h later FACS-sorted. Cells positive for VHS-GAT-GFP (A), unstransfected (B), or positive for GGA1-GFP (C) were reseeded, cultured overnight, pulsed for 5 min with [³⁵S]-sulfate, and chased for 15 min to allow budding from the TGN. PNS was prepared from both cell populations and fractionated on a sucrose VG, and an aliquot of each fraction was subjected to SDS–PAGE and autoradiography. The peak of ISGs is found in fraction 3 and 4 under all conditions.

VHS-GAT-GFP but not GGA1-GFP accumulate MPR at a juxtanuclear region and redistribute AP1 to the cytosol. PC12 cells transfected with VHS-GAT-GFP (A–D, I–J) or GGA1-GFP (E–H, L–N) were fixed 24 h post-transfection and labeled with (B, F) anti-MPR, anti-γ AP (J, M) Abs (red), or (C, G) anti-TGN38 (blue), and secondary Abs. Representative confocal images with the VHS-GAT-GFP (A, I) and GGA1-GFP (E, L) channels are shown. (D), (H), (K), and (N) are merged channels of (B) and (C, F and G, I and J, and L and M), respectively. MPR accumulation and AP1 redistribution can be observed in the VHS-GAT-GFP transfected cells (^*). Bars=2 μm.

VHS-GAT-GFP is found on ISGs, and inhibits removal of MPR from ISGs. PC12 cells transfected with (A) VHS-GAT-GFP (green) or (B) GGA1-GFP (green) were fixed after 24 h and labeled with anti-TGN38 (blue) and anti-SgII (red) Abs and secondary Abs. Channel merges are shown in bottom right of each panel. Insets in the merged channels are the boxed region magnified. Arrows, GFP and SgII-positive, but TGN38-negative structures. Bar=2 μm. (C–F) Fractions from EGs, which were loaded with (C, D) VG fractions 2–4, or (E, F) fractions 5–7 prepared from PNS of PC12 cells transfected with VHS-GAT-GFP (C, E) or GGA1-GFP (D, F). Proteins precipitated from the EG fractions were subjected to SDS–PAGE and immunoblotting using anti-SgII (upper panels) and anti-GFP (lower panels) Abs. Position of ISGs and MSGs on EGs is indicated. (G) ISG and MSG fractions prepared from [³⁵S]-Met/Cys-labeled FACS-sorted cells expressing VHS-GAT-GFP or GGA1-GFP were subjected to IP with anti-MPR ab, or a preimmune control (PI).

Inhibition of SgII processing in VHS-GAT-GFP- but not in GGA1-GFP-expressing cells. PC12 cells cotransfected with PC2 and either VHS-GAT-GFP (black lines in A, B, D, E), or as a negative control GGA1-GFP (red lines in A, B, D, E). At 24 h post–transfection, the cells were fixed and labeled with (A) and (C) anti-PC2 and anti-p18, (B) anti-SgII and anti-p18, (D) anti-PC2 and anti-Mann II, and (E) anti-PC2, and analyzed by flowcytometry with secondary Abs. Cells positive for either VHS-GAT-GFP (black) or GGA1-GFP (red) and PC2 (or p18 in B) were gated and analyzed for the distribution of p18 (A), SgII (B), MannII (D), and PC2 (E). Student's t-tests show that the ratios (median p18 in PC2-positive cells)/(median p18 in PC2-negative cells) in (A) and (median SgII in p18-positive cells)/(median SgII in p18-negative cells) in (B) are significantly (A) smaller (P⩽5%) or (B) larger (P⩽5%) in VHS-GAT-GFP- than in GGA1-GFP-positive cells. No significant difference was observed between the VHS-GAT-GFP and GGA1-GFP populations in (D) and (E). (C) Dot plots testing the correlation between p18 and PC2 levels in VHS-GAT-GFP- (black) and GGA1-GFP- (red) gated cells. One-way ANOVA shows that a significant (P⩽2.5%) linear trend exists between p18 and PC2 in GGA1-GFP-positive cells, but not in the VHS-GAT-GFP-positive cells. (F, G) PC12 cells were cotransfected with either VHS-GAT-GFP (F), or GGA1-GFP (G) and PC2 and fixed 24 h post-transfection. HA (red) and PC2 (blue) were visualized by confocal microscopy with secondary Abs, GFP channel (white). (H) PC12 cells cotransfected with PC2 and either GGA1-GFP (left lane), or VHS-GAT-GFP (right lane) were, after FACS sorting for GFP- and PC2-positive cells, lysed and subjected to SDS–PAGE and immunoblotting with anti-PC2 and anti-p18 Abs.

Depletion of GGA3 in PC12 cells inhibits VAMP4-Flag removal from maturing SGs and reduces pro-PC2 maturation. (A) PC12 cells were treated either with siControl (upper panel) or siGGA3 (lower panel), fixed, and labeled with anti-GGA3 Ab and secondary Ab (green). Representative confocal images, phase-contrast images, and channel merges are shown. Bar=20 μm. (B) Real-time RT–PCR analysis of GGA3 mRNA in PC12 cells transfected with siControl or siRNA against GGA3 (siGGA3). (C–E) PC12 cells were transfected with siControl (C) or siGGA3 (D), and 48 h later, retransfected with the siRNAs, and with CgB-HA and VAMP4-Flag constructs, and fixed 24 h later. Anti-GGA3 (white), anti-HA (green), and anti-Flag (red) Abs were visualized with secondary Abs by confocal microscopy. (E) Quantitation of the percentage of all VAMP4-Flag structures colocalizing with CgB-HA, n=3. Colocalization was quantified using the LSM software. Student's t-test shows that the extent of VAMP4-Flag colocalized with CgB-HA is significantly (P⩽5%) higher in siGGA3- than in siControl-treated cells. (F) PC12 cells were treated with siControl or siGGA3 and cotransfected with the PC2 plasmid. PC2-positive cells were FACS sorted from the siControl- and siGGA3-treated cell populations, and analyzed as in Figure 6F with PC2 and p18 Abs. The maturation of pro-PC2 to PC2 was inhibited in siGGA3-treated cells resulting in, on average, a 180% increase in the ratio (pro-PC2/mature PC2) in siGGA3- relative to siControl-treated cells. p18 levels were also reduced in siGGA3 cells by 30% on average.

VHS-GAT-GFP but not GGA1-GFP inhibits the removal of VAMP4 from maturing ISGs. (A, B) AtT20 cells were cotransfected with either (A) VHS-GAT-GFP or (B) GGA1-GFP and VAMP4-Flag and fixed 24 h post-transfection. Anti-Flag (red) and anti-ACTH (blue) were visualized with secondary Abs by confocal microscopy. GFP channel is shown in white. Arrow, ACTH and VAMP4-Flag colocalizing at cell tip. Bars=5 μm. (C, D) PC12 cells were cotransfected with CgB-HA and VAMP4-Flag and either (C) VHS-GAT-GFP or (D) GGA1-GFP, and fixed 24 h post-transfection. Anti-HA (red) and anti-Flag (blue) Abs were visualized with secondary Abs by confocal microscopy. White channel, GFP. Arrows in (C), VAMP4-Flag positive granules. Bars=1 μm. (E) A PNS from VHS-GAT-GFP and GGA1-GFP FACS sorted, [³⁵S]-Met/Cys-labeled cells was used to prepare ISG and MSG fractions. Membranes sedimented from these fractions were used for VAMP4 IP using a VAMP4 (V4) or an NR Ab. (F) Quantitation of the percentage of VAMP4-Flag structures colocalized with CgB-HA outside of the TGN/juxtanuclear area in PC12 cells transfected as in (C) (blue bar) and (D) (red bar). Percentage of VAMP4-Flag colocalization with CgB-HA was calculated based on the means of five independent observations, using the LSM 510 software. Student's t-test shows that the extent of colocalization is higher in the VHS-GAT-GFP-expressing cells than in cells expressing GGA1-GFP (P⩽1%). (G) Representative immuno-EM images of new granules positive for CgB-HA (10 nm gold, arrow) and VAMP4-Flag (5 nm gold, arrowhead). Images from VHS-GAT-GFP FACS-sorted cells are shown. (H) Quantitation of VAMP4-Flag-positive and -negative new granules in the two FACS-sorted populations. Fischer's exact test analysis shows the proportion of VAMP4-Flag-positive new granules in VHS-GAT-GFP cells is significantly higher than in GGA1-GFP cells (P⩽1%), n=3. Bar=100 nm.

A PHA-based ISG–ISG fusion assay shows that ISG–ISG fusion rate is not affected by VHS-GAT-GFP or GGA1-GFP overexpression. (A) The emission spectrum of a mixture of PHA-labeled ISGs, PNS, ATP-regenerating system, and GTP in FB (standard fusion conditions) was measured at 0 min (black), and after 20 min incubation (gray) at 37°C. After incubation, the PHA monomer fluorescence intensity (peaks at 377 and 397 nm) has increased. (B) Rates of fusion measured by the initial slope of a time-based monitoring of fluorescence, as shown in (C). Left panel, PHA-labeled ISGs were preincubated for 5 min on ice with PNS under standard fusion conditions (control), or with 25 U hexokinase and 3.3 mM glucose (ATP depletion). Following the preincubation, fluorescence was monitored over time as described in Materials and methods, and initial slopes calculated. Right panel, PHA-labeled ISGs were preincubated 10 min on ice under standard fusion conditions (as in left panel), or with 27 μg/ml Sx6 Ab, before monitoring fluorescence (n=3). Both ATP depletion and Sx6 Ab treatments caused a significant reduction in the initial slopes of fluorescence over time, as confirmed by Student's t-tests (P⩽1%). (C) A fusion assay performed using PHA-labeled ISGs in fusion buffer containing GTP- and ATP-regenerating system (dashed line), or with a PNS from 10⁷ FACS-sorted VHS-GAT-GFP-positive cells (black line), or 10⁷ FACS-sorted GGA1-GFP positive cells (gray line). The fluorescence of PHA monomers (ex. 330, em. 377) was recorded continuously at 37°C. Data shown are normalized to infinite dilution of PHA obtained by adding 1% Triton X-100 to each condition after the signal had reached a plateau. Fusion rates (averages of three experiments) as in (B) are shown in the right panel (n=3).