Fig 2: Changes in retrograde transport kinetics of CDMPR to the TGN upon silencing Vps26, Rab9a, or TIP47.(A, B, C, D) Cells stably expressing EGFP-CDMPR were transfected with non-targeting siRNA (control-kd) or with siRNA silencing expression of Vps26 (A) or Rab9a (C) as described in Fig 1. The cells were labeled with [35S]sulfate for up to 75 min in the presence of 2 µg/ml VHH-2xTS. The nanobodies were isolated by Ni/NTA beads and subjected to SDS-gel electrophoresis followed by Western-blot (WB) 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. Experiments as shown in panels (A) and (C) were quantified in panels (B) and (D), respectively, and presented as the percentage of the value of control-kd cells after 75 min (mean and SD of three independent experiments; two-sided t test: *P < 0.05; **P < 0.01). Control-kd is shown as black squares and target-kd as gray circles; uptake as open symbols, sulfation as filled symbols. (E, F) HeLa cells stably expressing His6/myc-tagged cathepsin D or PAUF were transfected with non-targeting control siRNA or RNAs silencing expression of Vps26 or Rab9a. Cells were incubated in serum-free medium supplemented with 5 mM mannose-6-phosphate for 2 h. Secreted procathepsin D (pCatD) or PAUF were collected by Ni/NTA beads and analyzed by immunoblotting with anti-myc antibodies. (G, H) Missorted procathepsin D or PAUF was quantified from immunoblots as shown in panels C and E, respectively, and normalized to the values of control knockdown cells (mean and SD of four [pCatD] and three [PAUF] independent experiments). (I, J) Cells stably expressing EGFP-CDMPR were transfected with non-targeting siRNA (control-kd) or with siRNA silencing expression of TIP47 and assayed and quantified as described above in panels (A, B, C, D) (mean and SD of three independent experiments).

Fig 3: Functionalized nanobodies to analyze retrograde transport of CDMPR upon silencing of adaptor protein-1, retromer, Rab9a, or TIP47.(A) Schematic representation of the functionalized nanobodies. The standard nanobody (VHH-std) consists of the GFP-specific VHH domain, T7 and HA epitope tags, a biotin acceptor peptide (BAP), and a hexahistidine (His6) purification tag. Other nanobodies in addition contain two tyrosine sulfation sequences (VHH-2xTS) or mCherry (VHH-mCherry). 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, T7, or mCherry epitopes, or with streptavidin-HRP (SA-HRP). Marker proteins with molecular weights in kilodalton are shown on the left. As previously reported (Buser et al, 2018; Buser & Spiess, 2019), mCherry-containing nanobodies are slightly susceptible to clipping between the VHH and mCherry domains. (C) HeLa cells were transfected with non-targeting siRNA or siRNAs targeting µ1A-adaptin, Vps26, TIP47, or Rab9a. 3 d after transfection, the cells were subjected to immunoblot analysis with antibodies against the indicated proteins. (D) To determine the knockdown (kd) efficiency, the residual protein was quantified in percent of the value after control-kd (mean and SD of three independent experiments). (E) HeLa cells stably expressing EGFP-CDMPR were depleted of µ1A-adaptin, Vps26, TIP47, or Rab9a as in (C). Cells were incubated for 1 h at 37°C with full medium containing 5 µg/ml VHH-mCherry (~0.1 µM), fixed, stained for EEA1 and nuclei (DAPI, blue), and imaged by fluorescence microscopy. Bar: 10 µm. (F) Quantitation of the percentage of cells displaying the CDMPR localization phenotypes “mainly TGN,” “mainly peripheral,” or “fully peripheral” as in Wassmer et al (2007) and Simonetti et al (2017). For each condition, random frames with a total of 136–140 cells were scored from three independent experiments. (G, H) Total and surface EGFP-CDMPR levels in RNAi-silenced cells were quantified by flow cytometry. Cells were incubated for 30 min at 4°C with VHH-mCherry for exclusive binding to EGFP-CDMPR at the cell surface, washed, dissociated and analyzed for GFP and mCherry fluorescence to determine the levels of total (G) and surface EGFP-CDMPR (H), respectively. Median fluorescence intensities above background of parental HeLa cells without EGFP-CDMPR of each condition were normalized to the average of cells treated with non-targeting control siRNA. For each condition, 50,000 cells were analyzed in each experiment (mean and SD of three independent experiments).

Fig 4: General tyrosine sulfation is not affected by silencing of retrograde machinery candidates.(A) HeLa cells stably expressing the sulfatable secretory protein a1-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).