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Huntingtin-associated protein 1 (HAP1) was initially identified as a binding partner of huntingtin, mutations in which underlie Huntington's disease. Subcellular localization and protein interaction data indicate that HAP1 may be important in vesicle trafficking, cell signalling and receptor internalization. In this study, a proteomics approach was used for the identification of novel HAP1-interacting partners to attempt to shed light on the physiological function of HAP1. Using affinity chromatography with HAP1-GST protein fragments bound to Sepharose columns, this study identified a number of trafficking-related proteins that bind to HAP1. Interestingly, many of the proteins that were identified by mass spectrometry have trafficking-related functions and include the clathrin light chain B and Sec23A, an ER to Golgi trafficking vesicle coat component. Using co-immunoprecipitation and GST-binding assays the association between HAP1 and clathrin light chain B has been validated in vitro. This study also finds that HAP1 co-localizes with clathrin light chain B. In line with a physiological function of the HAP1-clathrin interaction this study detected a dramatic reduction in vesicle retrieval and endocytosis in adrenal chromaffin cells. Furthermore, through examination of transferrin endocytosis in HAP1−/− cortical neurons, this study has determined that HAP1 regulates neuronal endocytosis. In this study, the interaction between HAP1 and Sec23A was also validated through endogenous co-immunoprecipitation in rat brain homogenate. Through the identification of novel HAP1 binding partners, many of which have putative trafficking roles, this study provides us with new insights into the mechanisms underlying the important physiological function of HAP1 as an intracellular trafficking protein through its protein-protein interactions. Refereed/Peer-reviewed

Huntingtin-associated protein-1 (HAP1) is involved in intracellular trafficking, vesicle transport, and membrane receptor endocytosis. However, despite such diverse functions, the role of HAP1 in the synaptic vesicle (SV) cycle in nerve terminals remains unclear. Here, we report that HAP1 functions in SV exocytosis, controls total SV turnover and the speed of vesicle fusion in nerve terminals and regulates glutamate release in cortical brain slices. We found that HAP1 interacts with synapsin I, an abundant neuronal phosphoprotein that associates with SVs during neurotransmitter release and regulates synaptic plasticity and neuronal development. The interaction between HAP1 with synapsin I was confirmed by reciprocal co-immunoprecipitation of the endogenous proteins. Furthermore, HAP1 co-localizes with synapsin I in cortical neurons as discrete puncta. Interestingly, we find that synapsin I localization is specifically altered in Hap1−/− cortical neurons without an effect on the localization of other SV proteins. This effect on synapsin I localization was not because of changes in the levels of synapsin I or its phosphorylation status in Hap1−/− brains. Furthermore, fluorescence recovery after photobleaching in transfected neurons expressing enhanced green fluorescent protein-synapsin Ia demonstrates that loss of HAP1 protein inhibits synapsin I transport. Thus, we demonstrate that HAP1 regulates SV exocytosis and may do so through binding to synapsin I. (Figure presented.) The Proposed mechanism of synapsin I transport mediated by HAP1 in neurons. HAP1 interacts with synapsin I, regulating the trafficking of synapsin I containing vesicles and/or transport packets, possibly through its engagement of microtubule motors. The absence of HAP1 reduces synapsin I transport and neuronal exocytosis. These findings provide insights into the processes of neuronal trafficking and synaptic signaling. Refereed/Peer-reviewed

Huntingtin-associated protein 1 (HAP1) was initially established as a neuronal binding partner of huntingtin, mutations in which underlie Huntington's disease. Subcellular localisation and protein interaction data indicate that HAP1 may be important in vesicle trafficking and cell signalling. In this study we establish that HAP1 is important in several steps of exocytosis in adrenal chromaffin cells. Using carbon-fibre amperometry we measured single vesicle exocytosis in chromaffin cells obtained from HAP1-/- and HAP1+/+ littermate mice. The number of Ca2+-dependent and Ca2+-independent full fusion events in HAP1-/- cells is significantly decreased compared to HAP1+/+ cells. We observe no change in the frequency of 'kiss-and-run' fusion events or in Ca2+ entry. While release per full fusion event is unchanged in HAP1-/- cells, early fusion pore duration is prolonged, as indicated by increased duration of pre-spike foot (PSF) signals. Kiss-and-run events have a shorter duration, indicating opposing roles for HAP1 in the stabilisation of the fusion pore during full fusion and transient fusion. We further demonstrate with electron microscopy a reduction in the number of vesicles docked at the plasma membrane of HAP1-/- cells and membrane capacitance measurements reveal a reduced size of the readily releasable pool of vesicles. Our study therefore illustrates that HAP1 regulates exocytosis by influencing the morphological docking of vesicles at the plasma membrane, the ability of vesicles to be released rapidly upon stimulation and the early stages of fusion pore formation. Refereed/Peer-reviewed