Journal: The FASEB Journal

Article Title: Role of ubiquitylation and USP8-dependent deubiquitylation in the endocytosis and lysosomal targeting of plasma membrane KCa3.1

doi: 10.1096/fj.11-187005

Figure Lengend Snippet: Membrane KCa3.1 is ubiquitylated during endocytic trafficking. A) At time 0 min, KCa3.1 is localized at the plasma membrane (red) and is completely endocytosed after 90 min incubation at 37°C. B) To detect ubiquitylated KCa3.1, plasma membrane channel was enzymatically biotinylated and labeled with streptavidin; cells at time 0 and 90 min were lysed in the presence of GST-TUBE2. Following pulldown, the immunoblot (IB) was probed using α-streptavidin Ab to identify BLAP-tagged KCa3.1/streptavidin. To control for nonspecific labeling, KCa3.1- expressing cells were labeled with streptavidin in the absence of enzymatic biotinylation (control lanes in all IB). No channel is detected in these controls, confirming the specificity of our labeling protocol. As shown in lane 3, endocytosed KCa3.1 is heavily ubiquitylated (time 90 min) compared with channel present at the plasma membrane (time 0 min; lane 2). Importantly, similar levels of KCa3.1 were detected for the two time points, as assessed by IB of total cell lysate (lanes 5 and 6). C) Samples were prepared and lysed as in B, then KCa3.1 was immunoprecipitated using streptavidin Ab, and the subsequent IB was probed using α-ubiquitin Ab. KCa3.1 is strongly ubiquitylated following endocytosis. D) HEK cells were doubly transfected with BLAP-KCa3.1 and HA-ubiquitin. Ubiquitin was immunoprecipitated using an anti-HA Ab and subsequently IB for streptavidin-tagged KCa3.1. E) KCa3.1 was immunoprecipitated using an anti-streptavidin antibody and the subsequent IB probed with anti-HA Ab.

Article Snippet: Antibodies Polyclonal α-streptavidin Ab was obtained from Genscript Corporation (Piscataway, NJ, USA).

Techniques: Membrane, Clinical Proteomics, Incubation, Labeling, Western Blot, Control, Expressing, Immunoprecipitation, Ubiquitin Proteomics, Transfection

Journal: The FASEB Journal

Article Title: Role of ubiquitylation and USP8-dependent deubiquitylation in the endocytosis and lysosomal targeting of plasma membrane KCa3.1

doi: 10.1096/fj.11-187005

Figure Lengend Snippet: Ubiquitylation is required for KCa3.1 endocytosis. A) BLAP-KCa3.1 was enzymatically biotinylated at the cell surface and labeled with streptavidin-Alexa555 or streptavidin and incubated at 37°C for 90 min in the absence or presence of hypertonic sucrose, an inhibitor of endocytosis, or the ubiquitin-activating enzyme (E1) inhibitor UBEI-41. Both hypertonic sucrose and UBEI-41 dramatically reduced the internalization of KCa3.1. B) GST-TUBE pulldown assay, followed by IB using α-streptavidin Ab, suggests that KCa3.1 may be ubiquitylated at the plasma membrane in the absence of endocytosis (compare lanes 5 and 2) and that inhibition of ubiquitylation blocks endocytosis of the channel (UBEI-41 treatment). Subsequent to endocytosis, KCa3.1 becomes heavily polyubiquitinated (T=90 min, lane 3).

Article Snippet: Antibodies Polyclonal α-streptavidin Ab was obtained from Genscript Corporation (Piscataway, NJ, USA).

Techniques: Labeling, Incubation, Ubiquitin Proteomics, Clinical Proteomics, Membrane, Inhibition

Journal: The FASEB Journal

Article Title: Role of ubiquitylation and USP8-dependent deubiquitylation in the endocytosis and lysosomal targeting of plasma membrane KCa3.1

doi: 10.1096/fj.11-187005

Figure Lengend Snippet: KCa3.1 is deubiquitylated before delivery to lysosomes. A) BLAP-KCa3.1 was enzymatically biotinylated and streptavidin labeled; the channel was allowed to internalize for the times indicated in the absence or presence of PR-619. PR-619 treatment significantly inhibited KCa3.1 degradation (bar graph, n=3). *P < 0.05. B) Cells were treated and labeled as in A, and the ubiquitylation of KCa3.1 was assessed by GST-TUBE pulldown assay. A representative blot is shown, indicating that PR-619 treatment prevents channel deubiquitylation. These data demonstrate that KCa3.1 needs to be deubiquitylated for proper lysosomal degradation.

Article Snippet: Antibodies Polyclonal α-streptavidin Ab was obtained from Genscript Corporation (Piscataway, NJ, USA).

Techniques: Labeling

Journal: The FASEB Journal

Article Title: Role of ubiquitylation and USP8-dependent deubiquitylation in the endocytosis and lysosomal targeting of plasma membrane KCa3.1

doi: 10.1096/fj.11-187005

Figure Lengend Snippet: DUB Chip as a tool to identify specific DUBs interacting with KCa3.1. A) BLAP-KCa3.1 was labeled with streptavidin-Alexa488 and incubated for the times indicated at 37°C. B) Cells were lysed in the presence of GST-TUBE2; the lysates were pulled down on GST beads, eluted, and hybridized on a DUB Chip (top panel). An interaction between fluorescently-tagged KCa3.1 and specific DUBs was quantified by measuring the fluorescence intensity (see Materials and Methods). DUB Chip data, expressed as relative fluorescence units (RFU), indicates an interaction between ubiquitylated KCa3.1 and both USP2 and USP8, and a weaker association with AMSH (bottom panel). C) Co-IP confirmed the interaction between KCa3.1 and USP8. Cells were cotransfected with myc-tagged KCa3.1 and either the WT or DN form of the GFP-USP8. USP8 was immunoprecipitated using either an anti-GFP Ab (lanes 1–3 and 5–6) or a nonspecific IgG (lanes 4 and 7) and subsequently IB for KCa3.1 with α-myc Ab. Total cell lysates are shown in lanes 8 and 9.

Article Snippet: Antibodies Polyclonal α-streptavidin Ab was obtained from Genscript Corporation (Piscataway, NJ, USA).

Techniques: Labeling, Incubation, Fluorescence, Co-Immunoprecipitation Assay, Dominant Negative Mutation, Immunoprecipitation

Journal: The FASEB Journal

Article Title: Role of ubiquitylation and USP8-dependent deubiquitylation in the endocytosis and lysosomal targeting of plasma membrane KCa3.1

doi: 10.1096/fj.11-187005

Figure Lengend Snippet: USP8 overexpression alters the ubiquitylation and degradation rate of KCa3.1 A) Cells were doubly transfected with BLAP-KCa3.1 and either GFP vector alone or the WT or DN GFP-USP8. KCa3.1 was labeled at the cell surface with streptavidin-Alexa555, and cells were incubated at 37°C for 8 h. In cells overexpressing either form of USP8, KCa3.1 degradation is slowed compared with control cells, as shown by the strong intracellular signal. Moreover, KCa3.1 clearly colocalizes with DN GFP-USP8 subsequent to endocytosis (bottom panel, arrow in overlay). B) Degradation rate of membrane KCa3.1 was quantified as above. Both WT and DN GFP-USP8 caused a significant delay in KCa3.1 degradation rate (bar graph, n=3). *P < 0.05. C) To correlate the degradation in B with the level of KCa3.1 ubiquitylation, cells were prepared as in B and lysed in the presence of GST-TUBE2 at the indicated times, followed by pulldown on GST beads and IB using α-streptavidin Ab. WT USP8 overexpression decreases ubiquitylation of KCa3.1, while DN USP8 prevents deubiquitylation, as compared with control cells.

Article Snippet: Antibodies Polyclonal α-streptavidin Ab was obtained from Genscript Corporation (Piscataway, NJ, USA).

Techniques: Over Expression, Transfection, Plasmid Preparation, Labeling, Incubation, Control, Membrane