Essential for the proteolytic processing of APP by BACE1 - and therewith for the generation of Abeta - is the transport and sorting of both proteins through endosomal and Golgi compartments. The family of Golgi-localized γ-ear-containing ARF-binding (GGA) proteins was shown to have striking functions in cargo sorting in these pathways. Recently, it was shown that GGA1 and GGA3 can interact with BACE1, that they are expressed in neurons and GGA3 is reduced in AD brain tissues. The GGA VHS-domain was found to be the major binding motif necessary for interaction with BACE1. We applied Immunoprecipitation and Fluorescence Lifetime imaging Microscopy (FLIM) with different GGA-domain deletions to analyze VHS-domain independent BACE1-GGA interaction and the binding capacity of the different deletion mutants. We further extended our approach by ELISA assays and Western Blot analysis to measure the influence on APP processing. Co-immunoprecipitation experiments showed that all GGA-VHS deletion-mutants can still be precipitated by BACE1, though to a lower extent than their wild type forms. Additionally, in FLIM experiments the lifetime of GGA-VHS deletion-mutants was only slightly increased compared to wild-type GGAs. Furthermore, cells co-transfected with APP, BACE and either wild-type GGAs or VHS deletion-mutants showed no significant differences in APP processing in Mesoscale Elisa assays and Western Blot analysis. Therefore, we extended our approach to identify other domains responsible for GGA-BACE1 interaction using several GGA domain-deletions and point-mutations to identify the major binding motif necessary for GGA-BACE interaction. In conclusion, our data suggest that the GGA VHS-domain has only limited influence upon the processing of APP and upon interaction with and trafficking of BACE1. As all other known GGA cargo proteins seem to be dependent on the VHS-domain differing BACE1 interactions are of high interest in the attempt to find BACE1 specific transport and trafficking modifiers.

Previous studies identified engulfment adapter phosphotyrosine binding (PTB) domain containing 1 (GULP1) as an NPXY-motif interactor of low-density lipoprotein receptor-related protein 1 (LRP1) and suggested a potential relevance in Alzheimer's disease (AD). Since AD associated proteins amyloid-β A4 precursor protein (APP) and LRP1 were shown to interact with the PTB domain of Fe65 and several other adapters via their intracellular NPXY-motifs, we examined a possible interaction of GULP1 PTB domain with the YENPTY-motif of APP. Here we demonstrate that GULP1 is present in human hippocampal and neocortical neurons. Confocal live cell imaging revealed that coexpressed and endogenous GULP1 colocalizes with APP in the Golgi and endoplasmic reticulum. Analysis of the interacting domains by co-immunoprecipitation of point and deletion mutants revealed that the interaction depends on the PTB domain of GULP1 and the YENPTY-motif of APP. Coexpression of GULP1 affected APP cell surface localization and suppressed generation of Aβ40/42 and sAPPα. Taken together, these data identify GULP1 as a novel neuronal APP interacting protein that alters trafficking and processing of APP.