Cooperative regulation of endocytic vesicle transport by yeast Eps15-like protein Pan1p and epsins

Nao Yoshida,Ippo Ogura,Makoto Nagano,Tadashi Ando,Junko Y Toshima,Jiro Toshima

doi:10.1016/j.jbc.2021.101254

Nao Yoshida, Ippo Ogura + Show 4 more

Open Access

https://doi.org/10.1016/j.jbc.2021.101254

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Abstract

Dynamic actin filaments are required for the formation and internalization of endocytic vesicles. Yeast actin cables serve as a track for the translocation of endocytic vesicles to early endosomes, but the molecular mechanisms regulating the interaction between vesicles and the actin cables remain ambiguous. Previous studies have demonstrated that the yeast Eps15-like protein Pan1p plays an important role in this interaction, and that interaction is not completely lost even after deletion of the Pan1p actin-binding domain, suggesting that additional proteins mediate association of the vesicle with the actin cable. Other candidates for mediating the interaction are endocytic coat proteins Sla2p (yeast Hip1R) and Ent1p/2p (yeast epsins), as these proteins can bind to both the plasma membrane and the actin filament. Here, we investigated the degree of redundancy in the actin-binding activities of Pan1p, Sla2p, and Ent1p/2p involved in the internalization and transport of endocytic vesicles. Expression of the nonphosphorylatable form of Pan1p, Pan1-18TA, caused abnormal accumulation of both actin cables and endocytic vesicles, and this accumulation was additively suppressed by deletion of the actin-binding domains of both Pan1p and Ent1p. Interestingly, deletion of the actin-binding domains of Pan1p and Ent1p in cells lacking the ENT2 gene resulted in severely defective internalization of endocytic vesicles and recruitment of actin cables to the site of endocytosis. These results suggest that Pan1p and Ent1p/2p cooperatively regulate the interaction between the endocytic vesicle and the actin cable.

Highlights

Cables, which are polarized linear bundles of actin filaments [8, 9], to mediate their transport to the early endosome [10, 11], but it still remains unclear how endocytic vesicles interact with such actin cables
We previously demonstrated that Pan1p directly mediates the interaction between endocytic vesicles and actin cables via its actin-binding activity [18]
We investigated the timing of actin cable recruitment to sites of endocytosis and demonstrated that 48% of actin cables appear at such sites before actin patch formation [28], implying clathrin-coated vesicles (CCVs) budding, whereas 39% of the cables appear thereafter

Summary

Introduction

Cables, which are polarized linear bundles of actin filaments [8, 9], to mediate their transport to the early endosome [10, 11], but it still remains unclear how endocytic vesicles interact with such actin cables. To examine the contribution of the actin-binding activities of these proteins to the formation of actin cable aggregates in the pan1-18TA mutant, we constructed the pan1-18TA or pan1-18TAΔABD mutant carrying an individual deletion of the THATCH/talinlike domain of Sla2p (Sla2ΔTHATCH) or deletion of the ACB domain of Ent1p (Ent1ΔACB) (Fig. 2A).

Results

Conclusion