The Sla1 adaptor‐clathrin interaction regulates coat formation and progression of endocytosis

Abstract

Clathrin-mediated endocytosis is a fundamental transport pathway that depends on numerous protein-protein interactions. Testing the importance of the adaptor protein-clathrin interaction for coat formation and progression of endocytosis in vivo has been difficult due to experimental constrains. Here, we addressed this question using the yeast clathrin adaptor Sla1, which is unique in showing a cargo endocytosis defect upon substitution of 3 amino acids in its clathrin-binding motif (sla1 ) that disrupt clathrin binding. Live-cell imaging showed an impaired Sla1-clathrin interaction causes reduced clathrin levels but increased Sla1 levels at endocytic sites. Moreover, the rate of Sla1 recruitment was reduced indicating proper dynamics of both clathrin and Sla1 depend on their interaction. sla1 cells showed a delay in progression through the various stages of endocytosis. The Arp2/3-dependent actin polymerization machinery was present for significantly longer time before actin polymerization ensued, revealing a link between coat formation and activation of actin polymerization. Ultimately, in sla1 cells a larger than normal actin network was formed, dramatically higher levels of various machinery proteins other than clathrin were recruited, and the membrane profile of endocytic invaginations was longer. Thus, the Sla1-clathrin interaction is important for coat formation, regulation of endocytic progression and membrane bending.

Protein context shapes the specificity of SH3 domain-mediated interactions in vivo

“…Sla1 compartmentalizes into foci at internalization sites on the plasma membrane where it is present until vesicles are fully internalized (Fig. 4C ); its deletion was shown to alter endocytosis dynamics 25 – 27 . The shuffling of Sla1 SH3s alters PPIs with partners involved in different phases of endocytosis (Fig.…”

“…During clathrin-mediated endocytosis, Sla1 is an adaptor linking cargos to clathrin and recruits members of the actin machinery via SH3-dependent PPIs (Fig. 4C ) 25 , 26 . Sla1 compartmentalizes into foci at internalization sites on the plasma membrane where it is present until vesicles are fully internalized (Fig.…”

Light-dependent inhibition of clathrin-mediated endocytosis in yeast

“…To investigate the effect of light-activated TL2 on endocytosis, we followed the cortical dynamics of the GFPtagged coat-associated protein Sla1-GFP. The yeast clathrin adaptor Sla1 is a component of the clathrin coat, whose spatiotemporal dynamics at endocytic sites has been well-characterized 12,13,33 . Spheroplasts of S. cerevisiae expressing Sla1-GFP were treated with either pre-activated TL2 (380 nm) or with vehicle, and cortical patches were live-imaged after 30 minutes incubation.…”

Protein context shapes the specificity of domain-peptide interactionsin vivo

“…During this process, vesicles are fully internalized following scission, before complex disassembly. It was previously reported that Sla1 deletion results in altered endocytosis dynamics and defective actin polymerisation (Holtzman, Yang and Drubin, 1993;Kaksonen, Toret and Drubin, 2005;Feliciano and Di Pietro, 2012;Tolsma, Cuevas and Di Pietro, 2018) . We found that Sla1 SH3 shuffling alters its interaction with partners that are themselves associated with different phases of endocytosis, such as clathrin (Chc1), Las17, Pan1, End3 and subunits of the Arp2/3 complex (ARC proteins) ( Figure 5C).…”

“…We extended these analyses by directly examining at the single-cell level Sla1's specific function in clathrin-mediated endocytosis ( Figure 5B) (Feliciano and Di Pietro, 2012;Mund et al , 2018;Tolsma, Cuevas and Di Pietro, 2018;Manenschijn et al , 2019) . During endocytosis, Sla1 plays a central role as an adaptor protein linking cargos to clathrin, in addition to participating in the recruitment of the actin machinery to internalization sites via SH3-dependent PPIs ( Figure 5B) (Feliciano and Di Pietro, 2012;Tolsma, Cuevas and Di Pietro, 2018) . Sla1 appears as compartmentalized into foci at the plasma membrane where it is present until cell membrane invagination is complete.…”