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. 2018 Aug 3:9:1070.
doi: 10.3389/fphys.2018.01070. eCollection 2018.

Golgin-97 Targets Ectopically Expressed Inward Rectifying Potassium Channel, Kir2.1, to the trans-Golgi Network in COS-7 Cells

Tarvinder K Taneja  1 , Donghui Ma  1 , Bo Y Kim  1 , Paul A Welling  1
Affiliations

Golgin-97 Targets Ectopically Expressed Inward Rectifying Potassium Channel, Kir2.1, to the trans-Golgi Network in COS-7 Cells

Tarvinder K Taneja et al. Front Physiol. .

Abstract

The inward rectifying potassium channel, Kir2.1, is selected as cargo at the trans-Golgi network (TGN) for export to the cell surface through a unique signal-dependent interaction with the AP1 clathrin-adaptor, but it is unknown how the channel is targeted at earlier stages in the secretory pathway for traffic to the TGN. Here we explore a mechanism. A systematic screen of Golgi tethers identified Golgin-97 as a Kir2.1 binding partner. In vitro protein-interaction studies revealed the interaction is direct, occurring between the GRIP domain of Golgin-97 and the cytoplasmic domain of Kir2.1. Imaging and interaction studies in COS-7 cells suggest that Golgi-97 binds to the channel en route through the Golgi. RNA interference-mediated knockdown of Golgin-97 prevented exit of Kir2.1 from the Golgi. These observations identify Golgin-97 as a Kir2.1 binding partner that is required for targeting the channel to the TGN. Based on our studies in COS-7 cells, we propose Golgi-97 facilitates formation of AP1-dependent export carriers for Kir2.1 by coupling anterograde delivery of Kir2.1 with retrograde recycling of AP-1 containing endosomes to the TGN.

Keywords: clathrin; golgi apparatus; inward rectifying K channel; membrane trafficking; potassium channel.

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Figures

FIGURE 1
FIGURE 1
Kir2.1 specifically binds to Golgin-97. Immunoprecipitation (IP) performed with (A) anti-Golgin-97; (C) anti-p230; (D) anti-GM130 antibodies from COS cells transfected with myc-Kir2.1 wild type (WT), the TGN-export mutant (Δ314–315), and the ER-export mutant (ΔFCY) and then immunoblotted (IB) with myc-antibodies to detect Kir2.1 in the Golgin immunoprecipitates. 5% input is shown below; (B) Quantification of the relative amounts of Kir2.1 immunoprecipatated with Golgin-97. ∗P < 0.05.
FIGURE 2
FIGURE 2
Golgin-97 GRIP domain directly interacts with Kir2.1. (A) Domain structure of Golgin-97, showing the GRIP domain and the coiled-coil domain. (B) Anti-myc immunoprecipitation (IP) followed by anti-HA immunoblotting (IB) from COS cells co-transfected with myc-Golgi 97 (Full length, Grip domain only, or coiled-coiled domain only) and HA-Kir2.1 wild type, WT, or the TGN-export mutant (Δ314–315) or (D) the HA-tagged Kir2.1 COOH-terminal cytoplasmic domain (CTCD). (C,E) Quantitative summaries comparing relative amounts of Kir2.1 (WT or Δ314–315) immunoprecipitated with full-length Golgin-97, or GRIP or Coiled-Coil domain, ∗P < 0.05. (F) GST pulldowns: GST alone or GST fusion proteins of the Kir2.1 CTCD were used to test for direct interaction with His-tagged GRIP domains of Golgin-97 or Golgin-245 purified from bacteria. Protein bound is shown relative to 5% input.
FIGURE 3
FIGURE 3
The Kir2.1 C-terminal cytoplasmic domain (CTCD) Interacts with Golgi-97 independently of ER/Golgi export and PDZ-binding signals. (A) Topological structure of Kir2.1, depicting C-terminal deletion mutants. (B) The HA-tagged Kir2.1 channel CTCD or deletion mutants, including Golgi-export Δ314/15 (aa181–426), ER export -FCY (aa181–372) or -PDZ (181–390), were co-transfected with myc-tagged Golgin-97, and cell lysates were subjected to immunoprecipitation anti-myc and then immunoblotted with anti-HA or anti-myc antibodies.
FIGURE 4
FIGURE 4
Mapping the residues in GRIP domain involved in Kir2.1 binding. (A) Model of the Golgin-97 GRIP domain. First helix directly interacts with Arl1, while the third helix contains residues (M733, Y740) that are necessary for binding to Kir2.1. (B) The HA-tagged Kir2.1 channel was co-transfected with myc-tagged Golgin-97 (WT or GRIP domain mutants), and cell lysates were subjected to immunoprecipitation with anti-myc and then immunoblotted with anti-HA to detect Kir2.1 bound. (C) Summary Data, Relative to Average WT Kir2.1 immunoprecipitated with wild type (WT) Golgin-97 GRIP. ∗P < 0.05.
FIGURE 5
FIGURE 5
Golgin-97 Interacts with Kir2.1 in the Golgi. (A) Confocal images of Kir2.1 (TGN-export mutant (Δ314–315) compared to ER-export mutant ΔFCY) in COS-cells (green) and Golgi-97 (red) before (top) and after 10 min of brefeldin A treatment. (B) Anti-Golgin-97 immunoprecipitation (IP) followed by anti-HA immunoblotting (IB) from COS cells transfected with the HA-Kir2.1 mutants and treated with vehicle (–) or Brefeldin (+).
FIGURE 6
FIGURE 6
Golgin-97 is required for efficient delivery of Kir2.1 to the cell surface membrane from the Golgi. (A) Immunoblot of Golgin-97 from COS cells transfected with scramble or Golgi-97 siRNA. (B) Surface Expression of external HA-epitope tagged Kir2.1 in COS cells co-transfected with scramble or Golgi-97 siRNA or nothing. Mock are cells transfected with vector alone. (C) Representative confocal microscopy of Kir2.1 in COS cells transfected with scrample, or Golgin-97 siRNA. (D) Rescue Studies, Surface Expression of external HA-epitope tagged Kir2.1 in COS cells co-transfected with scramble or Golgi-97 siRNA and Golgin-97 rescue cDNA (WT vs mutant), ∗P < 0.05.
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