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First published online November 3, 2003
doi: 10.1242/10.1242/jcs.00803

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Residues within the myristoylation motif determine intracellular targeting of the neuronal Ca2+ sensor protein KChIP1 to post-ER transport vesicles and traffic of Kv4 K+ channels

Dermott W. O'Callaghan*, Burcu Hasdemir*, Mark Leighton and Robert D. Burgoyne{ddagger}

The Physiological Laboratory, University of Liverpool, Crown Street, Liverpool L69 3BX, UK



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  		Fig. 1. Localisation of ECFP-KV4.2 within the secretory pathway. HeLa cells were transfected with plasmid encoding ECFP-Kv4.2. After fixation the cells were immunostained with anti-PDI (A-C), anti-ERGIC-53 (D-F), anti-ß-COP (G-I) or anti-{gamma}-adaptin (J-L) visualised with Texas Redstreptavidin and imaged by laser scanning confocal microscopy. Black and white images are shown in each case for ECFP-Kv4.2 and antibody staining as indicated in the overlaid images shown in colour (Kv4.2 in green and antibody staining in red with overlap seen in yellow). Scale bar: 10 µm.

 


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  		Fig. 2. Effect of KChIP1 on the localisation of ECFP-Kv4.2. HeLa cells were transfected with plasmids encoding ECFP-Kv4.2 and EYFP-Mem together (A-C) or in a triple transfection in combination with pcDNA3-KChIP1 (D-F). The localisation of ECFP-Kv4.2 and EYFP-Mem are shown individually and in a combined colour image (overlay; ECFP-Kv4.2 in green, EYFPMem in red) with co-localisation shown in yellow. Triple transfection resulted in translocation of ECFP-Kv4.2 to regions of the plasma membrane. Scale bar: 10 µm.

 


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  		Fig. 3. Localisation of ECFP-Kv4.2 in HeLa cells co-expressing NCS-1 (A-C), hippocalcin (D-F), KChIP1 (G-I) EYFP fusion proteins. HeLa cells were co-transfected with ECFP-Kv4.2 and the indicated EYFP fusion protein and single and combined colour images (overlay; Kv4.2 in green and the EYFP in red) are shown. Scale bar: 10 µm.

 


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  		Fig. 4. Differential localisation of KChIP1 and NCS-1 expressed in HeLa cells. In dual-transfected cells (A), NCS-1-ECFP showed a perinuclear localisation because of association with the TGN and a diffuse distribution because of plasma membrane association. In contrast, KChIP1-EYFP had a predominantly punctate localisation. The overlaid colour image shows KChIP1-EYFP in green and NCS-1-ECFP in red. (B) Following permeabilisation with digitonin KChIP1-EYFP remained mainly cell associated in the absence and presence of Ca2+. (C) KChIP1-EYFP localisation in HeLa cells during live cell imaging before and after 1- and 5-minute treatment with 3 µM ionomycin. Scale bar: 10 µm.

 


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  		Fig. 5. Localisation of KChIP1 requires myristoylation and a minimal myristoylation motif is sufficient. HeLa cells were transfected to express KChIP1-EYFP (A,C), KChIP1(G2A)-EYFP (B,D), KChIP1(1-11)-EYFP (E,F) or KChIP1-ECFP and KChIP1(1-11)-EYFP (G,H) and examined after 3 days (A-D, F-G) or 1 day (E). The cells in C and D were fixed and examined after permeabilisation with 1 µM digitonin for 15 minutes in the absence of added Ca2+. The images in G and H are from the same co-transfected cell after 3 days. Scale bar: 10 µm.

 


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  		Fig. 6. Hippocalcin(1-14) co-localises with NCS-1 but KChIP1(1-11) does not. Cells were transfected to express hippocalcin(1-14)-EGFP or KChIP1-EYFP along with NCS-1 either as a wild-type or ECFP-tagged construct. In these dual transfected cells the minimal myristoylation sequence from hippocalcin (hippocalcin (1-14)) targeted EGFP (B) to the same sites as NCS-1 detected by immunofluorescence (A) with complete overlap of localisation as shown in yellow in the overlaid image (C). In contrast, KChIP1(1-11)-EYFP (E) showed a distinct localisation to that of NCS-1-ECFP (D,F). The colour overlays show NCS-1 in green and the other constructs in red. Scale bar: 10 µm.

 


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  		Fig. 7. Localisation of NCS-1, KChIP1 and Kv4.2 in PC12 cells. PC12 cells were transfected to express NCS-1-ECFP (A), hippocalcin(1-14)-EGFP (B), KChIP1-EYFP (C), KChIP1(1-11)EYFP (D), ECFP-Kv4.2 (E) or ECFP-Kv4.2 along with KChIP1-EYFP (F). KChIP1-EYFP and KChIP1(1-11)-EYFP showed more punctate intracellular staining than NCS-1-ECFP and hippocalcin(1-14)-EGFP and the latter constructs that were also localised to the plasma membrane. For the cells co-expressing ECFP-Kv4.2 and KChIP1-EYFP only the image for ECFP-Kv4.2 is shown in F but essentially complete overlap of the two constructs was observed. Scale bar: 10 µm.

 


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  		Fig. 8. Effect of mutations within the myristoylation motifs of hippocalcin and KChIP1 on intracellular targeting. (Top) N-terminal sequences of hippocalcin and KChIP1 showing the mutations introduced and the positions of the conserved basic residues in NCS-1 and hippocalcin (arrows). (A-H) Localisation of minimal myristoylation sequences with the indicated mutations is shown after expression of EGFP (hippocalcin) or EYFP (KChIP1) fusion proteins in HeLa cells. The localisation of KChIP1(A3K,G6S, T7K, S9R)-EYFP was examined in cells co-transfected to also express NCS-1-CFP and the localisation of each fusion protein is shown individually and in an overlaid colour image where co-localisation is seen in yellow. Scale bar: 10 µm.

 


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  		Fig. 9. Quantification of the effect of KChIP1 on ECFP-Kv4.2 traffic to the plasma membrane and requirement for correct targeting of KChIP1. (A) HeLa cells were transfected to express ECFP-Kv4.2 alone or in combination with KChIP1-EYFP or KChIP1(G2A)EYFP as indicated. ECFP-Kv4.2 fluorescence was imaged and quantified by drawing around regions of interest on the outside and the inside of the plasma membrane to allow determination of the percentage of total fluorescence at the plasma membrane. (B) Mean data±s.e.m. derived from 25 cells expressing ECFP-Kv4.2 alone and 47 and 24 co-expressing KChIP1-EYFP or KChIP1(G2A)-EYFP, respectively. (C) HeLa cells transfected to co-express KChIP1(A3K,G6S,T7K,S9R)-EYFP (KChIPmut) and ECFP-Kv4.2 did not show cell surface expression of Kv4.2. The overlaid colour image shows Kv4.2 in red and KChIP1(A3K,G6S,T7K,S9R)-EYFP in green with co-localisation in yellow. Scale bar: 10 µm.

 


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  		Fig. 10. Comparison of the localisation of KChIP1 and markers for the transitional ER and ERGIC compartment. HeLa cells were transfected to express KChIP1-EYFP or KChIP1(1-11)-EYFP, fixed, immunostained with the indicated antibodies visualised with Texas Red-streptavidin and imaged by laser scanning confocal microscopy. HeLa cells in the bottom row were transfected with KChIP1(1-11)EYFP and ECFP-Kv4.2. Black and white images are shown in each case as indicated with overlaid images shown in colour with KCHIPEYFP or KChIP1(1-11)-EYFP in green and antibody staining or ECFP-Kv4.2 in red and overlap seen in yellow. Scale bar: 10 µm.

 

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© The Company of Biologists Ltd 2003