Figure 1.
Podocin localizes to endosomal vesicles. Immunofluorescence for transiently expressed, V5- (a–c, e) or Flag-tagged (d) podocin using anti-V5 or anti-Flag antibody in HeLa cells.
Costainings of endogenous calnexin (a), endogenous golgin-97 (b), endogenous EEA1 (c), Lysotracker Red (d) and eGfp-tagged and transiently expressed CD63 (e) as markers for the endoplasmatic reticulum, Golgi apparatus, early endosomes, acidic organelles and late endosomes respectively, displayed that podocin localizes to the endosomal compartment. Analogously to HeLa cells, transiently expressed V5-tagged podocin colocalizes with eGFP tagged CD63/LAMP3 in cultured human podocytes (f).
Figure 2.
A C-terminal domain regulates plasma membrane localization of podocin.
A. Immunofluorescence for a transiently expressed, Flag-tagged truncation of podocin (podocin1–285) reveals increased plasma membrane localization in comparison with podocin wild type in HeLa-cells (b and a respectively). B. Schematic representation of constructs consisting of the extracellular domain of CD16 and the transmembrane domain of CD7 fused to different parts of podocin. C. Immunofluorescence using anti-CD16 antibody showed primarily membranous staining patterns for CD-16-7-* and CD16-7-podocin1–285 (a, b). In contrast, immunofluorescence for CD16-7-podocin286–385 revealed multiple vesicular structures (c), thereby proving analogous localization of CD16-fusion constructs and V5/Flag-tagged constructs.
Figure 3.
The domain podocin340–350 regulates the subcellular localization of podocin.
A–D. Various truncations of Flag-tagged podocin were coexpressed with eGfp-tagged CD63 in HeLa-cells. Immunofluorescence using anti-Flag antibody revealed a primarily membranous staining pattern for podocin1–285 and podocin1–340 (a and b). In contrast, podocin1–350 and podocin wild type were shown to also localize to intracellular vesicles costaining with CD63 hinting at a crucial role of podocin340–350 in determining the subcellular localization of podocin (c and d).
Figure 4.
PodocinTVV339, 340,341 regulates the surface expression of podocin.
A. Amino acid-triplets within podocin335–350 were mutated using a Quickchange approach. Mutated plasmids were transiently expressed in HeLa cells and localization of mutants was assessed by immunofluorescence using anti-Flag antibody. In contrast to the other mutants, podocinTVV339,340,341AAA was shown to localize in a predominantly membranous pattern similar to podocin1–285 (a–d). B. Immunofluorescence of Flag-tagged podocin wild type, podocin1–285 and podocinTVV339, 340,341AAA confirmed localization in transgenic differentiated podocytes to be analogous to HeLa cells (a–c). C. 293T-cells were transfected with plasmids expressing either CD16-7-*, CD16-7-podocin286–385 or CD16-7-podocin286–385 TVV339,340,341AAA and cell surface expression was analyzed by FACS. As transfected cells also expressed Gfp driven from an internal ribosomal site from the same vector, gates were set to include Gfp-positive cells only. Cell surface expression of CD16-7-* and CD16-7-podocin286–385TVV339,340,341AAA, was comparable (b), while less CD16-7-podocin286–385 could be detected at the plasma membrane, consistent with a role of podocinTVV339,340,341 in regulating internalization of podocin (a).
Figure 5.
PodocinTVV339,340,341 regulates the turnover of podocin through a lipid raft-independent mechanism.
A. Differentiated podocytes stably expressing Flag-tagged podocin, podocin1–285 or podocinTVV339,340,341AAA were exposed to the translation inhibitor cycloheximide for the times as indicated and analyzed per western blot using anti-Flag antibody (a). Actin levels detected by anti-actin antibody served as loading control. Podocin1–285 and podocinTVV339,340,341AAA were shown to be more stable than podocin wild type, consistent with a regulatory role of podocinTVV339,340,341 in its degradation. (b) Summarizes the results of three experiments. Podocin levels were normalized to actin levels. B. (a) shows a schematic comparison between the PHB-domain proteins podocin and stomatin. Umlauf et al. proved a motif partially overlapping with podocinTVV339,340,341 to play a crucial role in lipid raft binding. (b) HEK293T cells were transfected with the plasmids as indicated, lysed in 1% TX-100 on ice and subjected to flotation gradient centrifugation to prepare detergent-resistant membranes (DRM). In contrast to the control protein transferrin receptor, both podocin wild type and podocinTVV339,340,341AAA were detected in DRM. C. Graphical representation of the structure prediction analysis of podocin using the I-Tasser algorithm revealed exposed position of podocinTVV339, 340,341.