# Molecular basis for assembly and activation of the Hook3 − KIF1C complex-dependent transport machinery

## TL;DR

This study reveals how Hook3 and KIF1C form a complex to enable anterograde cargo transport in cells, providing insights into the molecular mechanisms of microtubule-based transport.

## Contribution

The study identifies the structural and functional basis for the Hook3-KIF1C complex and its role in cargo transport.

## Key findings

- Hook3 and KIF1C form a 2:2 heterotetrameric complex essential for anterograde transport.
- KIF1C competitively interacts with PTPN21 and Hook3, promoting its activation.
- Interaction with KIF1C is necessary for Hook3-mediated transport in RPE1 cells.

## Abstract

Microtubule-associated cargo transport, a central process governing the localization and movement of various cellular cargoes, is orchestrated by the coordination of two types of motor proteins (kinesins and dyneins), along with diverse adaptor and accessory proteins. Hook microtubule tethering protein 3 (Hook3) is a cargo adaptor that serves as a scaffold for recruiting kinesin family member 1C (KIF1C) and dynein, thereby regulating bidirectional cargo transport. Herein, we conduct structural and functional analyses of how Hook3 mediates KIF1C-dependent anterograde cargo transport through interaction with KIF1C and PTPN21. We verify the interactions among the three proteins and determine the crystal structure of the Hook3(553–624) − KIF1C(714–809) complex. Subsequent structure-based mutational analysis demonstrates that this complex formation is necessary and sufficient for the interaction between the full-length proteins in HEK293T cells and plays a key role in Hook3- and KIF1C-mediated anterograde transport in RPE1 cells. Thus, this study provides a basis for a comprehensive understanding of how Hook3 cooperates with other components during the initial steps of activation and assembly of the Hook3- and KIF1C-dependent cargo transport machinery.

Hook3 and PTPN21 sequentially and competitively interact with KIF1C, promoting its activation and facilitating KIF1C-dependent cargo transport. This study reveals how Hook3 serves as a mechanical platform for assembling cargo transport machinery.

Hook3 forms a 2:2 heterotetrameric complex with KIF1C.KIF1C competitively interacts with PTPN21 and Hook3.Interaction with KIF1C is necessary for Hook3-mediated anterograde cargo transport from the cytoplasm to the cell tips in RPE1 cells.

Hook3 forms a 2:2 heterotetrameric complex with KIF1C.

KIF1C competitively interacts with PTPN21 and Hook3.

Interaction with KIF1C is necessary for Hook3-mediated anterograde cargo transport from the cytoplasm to the cell tips in RPE1 cells.

Hook3 and PTPN21 sequentially and competitively interact with KIF1C, promoting its activation and facilitating KIF1C-dependent cargo transport. This study reveals how Hook3 serves as a mechanical platform for assembling cargo transport machinery.