Adaptor Protein Complex Interaction Map in Arabidopsis Identifies P34 as A Common Stability Regulator

Introduction

Transport and distribution of biomolecules between plant cell endomembrane systems are essential for plant growth and development. Adaptor Protein (AP) is an evolutionarily very conserved class of complexes that are involved in the fine regulation of endosomal transport in plants. Through evolutionary analysis and functional studies, five families of junctional proteins from AP-1 to AP-5 and the TPLATE protein complex have been reported in Arabidopsis. Among them, AP-2 and TPLATE complexes are precisely involved in endocytosis in plants, while AP-1, AP-3 and AP-4 play important roles in endosomal transport initiated by the trans-Golgi reticulum (TGN). However, the precise composition of the AP-5 complex and its function have not been elaborated, while the regulatory processes of the auxiliary interacting proteins and targeting transport proteins of the plant junctional protein complex are also unknown.

Methods

To investigate the precise composition of plant junctional protein complexes, a series of proteomics, protein purification, and mass spectrometry experiments were performed using Arabidopsis suspension cells expressing specific decoy proteins, respectively. To further elucidate the functions of plant junction proteins and their interaction networks, CoIP, BiFC, CRISPR-mediated gene knockdown, and immunoblot analysis were performed, respectively.

Results

Using Arabidopsis suspension cells expressing specific decoy proteins, a series of proteomic purification methods such as tandem affinity purification (TAP), single-step affinity purification (AP-MS) and neighboring protein labeling (PL-MS) combined with mass spectrometry were used to construct a total of 536 proteins and 926 pairs of interacting AP complex interactions, while new core proteins such as BAG4, AAK1 and P34 interacting with multiple AP complexes were identified.

Fig. 1 Interacting networks of splice protein complexes.

The research team has further validated the interaction network of BAG4, AAK1 and P34 by using CoIP and BiFC, and has precisely elucidated the composition of the AP-5 complex, identifying new subunits SPG11 and SPG15 in the AP-5 complex in plant cells. The functional aggregation analysis revealed that the AP complex may be involved in other biological functions such as mRNA transport and cell division, which is important for a comprehensive understanding of the function of the AP complex.

Since P34 interacts with both AP-1, AP-2 and AP-4, the research team further explored its biological function. Knockdown of P34 gene by CRISPR revealed that p34 pure mutation caused embryonic lethality. Embryonic developmental experiments showed that P34 knockout mutations delayed embryonic development and affected final maturation of the embryo. Further, knockdown of P34 by inducible CRISPR was found to affect the growth and weight orientation of plant primary roots, while AP-1, AP-2 and AP-4 proteins were found to be extremely unstable in the p34 mutant by immunoblot analysis. In order to investigate whether P34 affects the endosomal transport process regulated by the reciprocal AP complex, the team first examined the endocytosis process involved in the regulation of AP-2 and found that the uptake of FM4-64 dye and the transport of PIN2 regulated by endocytosis were differentially impaired in the p34 mutant. In addition, the Golgi-localized proteins SYP61 and MTV1, which depend on AP-1 and AP-4, respectively, showed similar distributions in the p34 mutant as in ap1 and ap4, whereas AP-3-associated translocation was not significantly affected in the p34 mutant.

Fig. 2 P34-regulated transport in the endosomal system.

In summary, P34 has an important and specific role in the protein stability of AP-1, AP-2, and AP-4 and the associated endosomal transport. Interestingly, in Arabidopsis AP-1, AP-2 and AP-4 are all AP complexes that interact with lattice proteins3, so it is very interesting direction to explore whether the function of P34 is related to lattice proteins in the future.

Summary

This study reports the protein interaction network of the Arabidopsis junction protein (AP) complex, which provides an important research resource for understanding the regulatory network of the plant endosomal transport system, and reveals that P34 plays a central stabilizing factor in multiple AP complexes.

Reference:

Wang, P.; et al. Adaptor protein complex interaction map in Arabidopsis identifies P34 as a common stability regulator. Nature Plants, 2023: 1-17.

* For research use only. Not intended for any clinical use.

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Adaptor Protein Complex Interaction Map in Arabidopsis Identifies P34 as A Common Stability Regulator

Introduction

Methods

Results

Summary

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