Secretory carrier membrane proteins interact and regulate trafficking of the organellar (Na+,K+)/H+ exchanger NHE7
- PMID: 15840657
- DOI: 10.1242/jcs.02315
Secretory carrier membrane proteins interact and regulate trafficking of the organellar (Na+,K+)/H+ exchanger NHE7
Abstract
The mammalian (Na(+),K(+))/H(+) exchanger NHE7 resides chiefly in the trans-Golgi network (TGN) and post-Golgi vesicles where it is thought to contribute to organellar pH homeostasis. However, the mechanisms that underlie the targeting and regulation of NHE7 are unknown. To gain insight into these processes, yeast two-hybrid methodology was used to screen a human brain cDNA library for proteins that interact with the cytoplasmic C-terminus of NHE7. One binding partner we identified was SCAMP2, a member of the secretory carrier membrane protein (SCAMP) gene family. Direct association of these two proteins was further supported by co-immunolocalization and co-immunoprecipitation analyses using transfected cells, by their co-sedimentation in membrane fractions resolved on sucrose density gradients, and by in vitro protein binding assays. Other members of the SCAMP family, such as SCAMP1 and SCAMP5, also associated with NHE7. The majority of the NHE7-SCAMP complexes accumulated at the TGN, but a minor fraction also resided in recycling vesicles. Biochemical analyses indicated that the C-terminal cytoplasmic tail of NHE7 bound preferentially to a highly conserved cytoplasmic loop between the second and the third transmembrane segments (TM2-TM3 loop) of SCAMP2. A deletion mutant of SCAMP2 lacking this region (SCAMP2/Delta184-208) bound weakly to NHE7, but caused a significant fraction of NHE7 and wild-type SCAMP2 to redistribute to a pool of scattered recycling vesicles without noticeably affecting the location of other resident TGN (syntaxin 6) or Golgi cisternae (GM130) proteins. Conversely, a GFP-tagged TM2-TM3 construct of SCAMP2 interacted with NHE7, but also led to the redistribution of NHE7 to dispersed vesicular structures. We propose a model wherein SCAMPs participate in the shuttling of NHE7 between recycling vesicles and the TGN.
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