Abstract
Huntingtin-associated protein-1 (HAP1) was initially identified as an interacting partner of huntingtin, the Huntington disease protein. Unlike huntingtin that is ubiquitously expressed throughout the brain and body, HAP1 is enriched in neurons, suggesting that its dysfunction could contribute to Huntington disease neuropathology. Growing evidence has demonstrated that HAP1 and huntingtin are anterogradely transported in axons and that the abnormal interaction between mutant huntingtin and HAP1 may impair axonal transport. However, the exact role of HAP1 in anterograde transport remains unclear. Here we report that HAP1 interacts with kinesin light chain, a subunit of the kinesin motor complex that drives anterograde transport along microtubules in neuronal processes. The interaction of HAP1 with kinesin light chain is demonstrated via a yeast two-hybrid assay, glutathione S-transferase pull down, and coimmunoprecipitation. Furthermore, HAP1 is colocalized with kinesin in growth cones of neuronal cells. We also demonstrated that knocking down HAP1 via small interfering RNA suppresses neurite outgrowth of PC12 cells. Analysis of live neuronal cells with fluorescence microscopy and fluorescence recovery after photobleaching demonstrates that suppressing the expression of HAP1 or deleting the HAP1 gene inhibits the kinesin-dependent transport of amyloid precursor protein vesicles. These studies provide a molecular basis for the participation of HAP1 in anterograde transport in neuronal cells.
Highlights
Huntingtin-associated protein-1 (HAP1)2 was the first protein identified to interact with huntingtin, the Huntington disease (HD) protein [1, 2]
Yeast two-hybrid analysis revealed that a C-terminal fragment of HAP1 present in both rodent isoforms binds kinesin light chain-2 (KLC-2, Fig. 1), a subtype of KLC that is highly homologous to kinesin light chain-1 (KLC-1) with 71.1% amino acid identity [30]
The findings of the present study provide a biochemical basis for the involvement of HAP1 in kinesin-based anterograde transport
Summary
Huntingtin-associated protein-1 (HAP1) was the first protein identified to interact with huntingtin (htt), the Huntington disease (HD) protein [1, 2]. It is believed that the expanded polyQ confers an abnormal protein conformation and affects the function of other neuronal proteins [3] This idea, or the theory of gain of function, is strongly supported by the fact that polyQ expansion causes htt to abnormally interact with other proteins [4, 5]. HAP1 might be involved in endocytotic trafficking of membrane receptors Evidence to support this idea includes the interaction of HAP1 with Hrs, which plays a critical role in the endocytosis of epidermal growth factor receptor [10]. We demonstrate that HAP1 interacts with the kinesin motor complex and that suppressing the expression of HAP1 inhibits kinesin-associated transport in neurons, providing the biochemical basis for the involvement of HAP1 in anterograde transport in neuronal cells
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