Fig 2: General tyrosine sulfation is not affected by silencing of retrograde machinery candidates.(A) HeLa cells stably expressing the sulfatable secretory protein α1-protease inhibitor (or anti-trypsin; SHMY-A1Pi; S, signal sequence of hemagglutinin; H, His6 epitope; M, myc epitope; Y, TS site) were transiently depleted of TIP47, Rab9a, Vps26, μ1A-adaptin, epsinR, and GGAs (GGA1–3) for 72 h. Cells were labeled with [35S]sulfate labeling for 75 min, lysed, HMY-A1Pi was isolated by Ni/NTA beads, and subjected to SDS-gel electrophoresis followed by autoradiography ([35S]). In parallel, aliquots of the cell lysates were immunoblotted for actin as a control for the amount of cells used. (B) Quantitation of total tyrosine sulfation as judged by sulfation of the secretory reporter HMY-A1Pi as in panel (A) is shown in percent of the value present in control-kd (mean and SD of three independent experiments).

Fig 3: Effect of rapid GGA2 inactivation on retrograde and anterograde CDMPR transport using derivatized anti-mCherry nanobodies.(A, D) Schematic outline of the retrograde (A) anterograde (D) transport sulfation assays, respectively, with HeLa-GGA2ks/mCherry-CDMPR cells. (B) HeLa-GGA2ks cells stably expressing mCherry-CDMPR were siRNA-silenced for endogenous GGA1–3. The cells were starved for sulfate for 1 h, and then labeled for up to 75 min with [35S]sulfate in the presence of LaM4-3xTS nanobodies either in the absence (−) or presence of 500 nM rapamycin to inactivate GGA2-FKBP (+Rapa). The nanobodies were isolated by Ni/NTA beads and subjected to SDS-gel electrophoresis followed by immunoblot analysis (anti-His6) and autoradiography ([35S]). In parallel, aliquots of the cell lysates were immunoblotted for actin as a control for the amount of cells used. (C) Experiments as shown in panel B were quantified and presented as the percentage of the value in the absence of rapamycin after 75 min (mean and SD of three independent experiments). Without rapamycin is shown as black squares, with rapamycin as gray circles; uptake as open symbols, sulfation as filled symbols. (E) HeLa-GGA2ks cells stably expressing mCherry-CDMPR were siRNA-silenced for endogenous GGA1–3, followed by starvation for sulfate in the presence of LaM4-3xTS to preload mCherry-CDMPR in the surface/endosome/TGN pool. The cells were then labeled with [35S]sulfate for up to 75 min in the continued presence of LaM4-3xTS, without or with addition of 500 nM rapamycin after 15 min (arrow) to inactivate GGA2 (+Rapa). (B) Nanobody analysis was performed as above (B). (F) Experiments as shown in panel E were quantified and presented as the percentage of the value in the absence of rapamycin after 75 min (mean and SD of three independent experiments). Without rapamycin is shown as black squares, with rapamycin as gray circles; uptake as open symbols, sulfation as filled symbols.

Fig 4: Cell surface levels of EGFP-CDMPR is not altered by silencing of retrograde machinery candidates.HeLa cells stably expressing EGFP-CDMPR were transiently transfected with non-targeting siRNA (Control-kd) or siRNAs targeting TIP47, Rab9a, Vps26, μ1A, epsinR, GGA1–3, AP-2α, or clathrin CHC17. 3 d after transfection, cells were incubated with 2 μg/ml VHH-2xTS in PBS at 4°C for 1 h to label the surface fraction of EGFP-CDMPR. Subsequently, cells were washed and lysed, and endogenous protein levels were analyzed by immunoblotting with antibodies against the indicated proteins (VHH-2xTS was detected with anti-His6). Only knockdown of CHC17 influenced surface levels of EGFP-CDMPR (monitored by bound nanobody). Total EGFP-CDMPR remained unaffected in all knockdowns.

Fig 5: Derivatized anti-mCherry nanobodies for use with GGA2 knocksideways cells.(A) Schematic representation of the functionalized anti-mCherry nanobodies. The standard nanobody (LaM4-std) consists of the mCherry-specific LaM4 domain, T7 and HA epitope tags, a biotin acceptor peptide (BAP), and a hexahistidine (His6) purification tag. Other nanobodies in addition contain one to three tyrosine sulfation sequences (TS). Scale bar in aa. (B) Bacterially expressed and purified nanobodies (30 μg) were analyzed by SDS-gel electrophoresis and Coomassie staining (left). Immunoblot analysis of nanobodies (10 ng) with antibodies against the HA, His6, or T7, or with streptavidin-HRP (SA-HRP). Marker proteins with molecular weights in kilodalton are shown on the left. (C) Parental HeLaM cells and HeLaM-GGA2ks cells stably expressing mCherry-CDMPR were transfected with non-targeting siRNA (−) or siRNAs silencing endogenous GGA1–3 (+). Cell lysates were subjected to immunoblot analysis with antibodies against the indicated proteins. (D) HeLaM-GGA2ks cells stably expressing mCherry-CDMPR were transfected with siRNAs targeting endogenous GGA1–3. These cells were transfected with a plasmid expressing His6/myc-tagged procathepsin D 24–36 h before analysis. Media of cells incubated for 2 h in serum-free medium supplemented with 5 mM mannose-6-phosphate to prevent cathepsin D binding to surface MPRs, and with or without rapamycin (+ or − Rapa, respectively) were analyzed by collecting procathepsin D (pCatD) with Ni/NTA beads and immunoblotting with anti-myc antibodies. Cell lysates were immunoblotted for actin as a control. (E) Procathepsin D missorted upon knocksideways of GGA2 (+Rapa) was quantified from immunoblots as shown in panel (D), normalized to the DMSO-treated (−Rapa) control (mean and SD of four independent experiments). (F) HeLa-GGA2ks cells stably expressing mCherry-CDMPR after silencing endogenous GGA1–3 were treated with or without 500 nM rapamycin for 1 h and processed for fluorescence microscopy to detect mCherry-CDMPR and recombinant GGA2-FKBP. Bar: 10 μm.