The AP‐2 complex is required for proper temporal and spatial dynamics of endocytic patches in fission yeast

Abstract

In metazoans the AP-2 complex has a well-defined role in clathrin-mediated endocytosis. By contrast, its direct role in endocytosis in unicellular eukaryotes has been questioned. Here, we report co- immunoprecipitation between the fission yeast AP-2 component Apl3p and clathrin, as well as the genetic interactions between apl3Δ and clc1 and sla2Δ/end4Δ mutants. Furthermore, a double clc1 apl3Δ mutant was found to be defective in FM4-64 uptake. In an otherwise wild-type strain, apl3Δ cells exhibit altered dynamics of the endocytic sites, with a heterogeneous and extended lifetime of early and late markers at the patches. Additionally, around 50% of the endocytic patches exhibit abnormal spatial dynamics, with immobile patches and patches that bounce backwards to the cell surface, showing a pervasive effect of the absence of AP-2. These alterations in the endocytic machinery result in abnormal cell wall synthesis and morphogenesis. Our results complement those found in budding yeast and confirm that a direct role of AP-2 in endocytosis has been conserved throughout evolution.

Traffic Through the Trans-Golgi Network and the Endosomal System Requires Collaboration Between Exomer and Clathrin Adaptors in Fission Yeast

“…cfr1 + exhibits genetic interaction with end4 + , suggesting a possible exomer role in endocytosis. Although the efficient FM4-64 uptake in cfr1D does not solely contradict this hypothesis because mutants in clathrin and AP-2 are able to internalize this dye (de León et al 2013(de León et al , 2016, this observation, together with the fact that Cfr1 is not observed at the PM (Figure 1), suggests that this function might be exerted after cargo internalization. It is possible that AP-2 contacts exomer at endosomes.…”

“…Western blots to detect soluble proteins were performed as described (Sharifmoghadam and Valdivieso 2009;de León et al 2016). To detect membrane proteins, the cleared lysates were incubated in the presence of 1.6 M urea in a tube rotator for 16 hr at 4°and the proteins were denatured at 65°for 5 min.…”

“…Finally, the existence of genetic interaction between exomer and clathrin was examined. The clc1-232 point mutant was used to perform the analysis because the clc1D mutant is lethal unless cultivated with an osmotic stabilizer (de León et al 2013(de León et al , 2016. clc1-232 exhibits synthetic growth defects when combined with apl3D, apm1D, and gga22D mutations (de León et al 2016 and Figure 5D).…”

The AP-2 complex has a specialized clathrin-independent role in apical endocytosis and polar growth in fungi

“…In a more recent study it has been shown that clathrin contributes to the regularity of vesicle scission and vesicle size, but is not required for elongating or shaping the endocytic membrane invagination, but again the role of AP-2 was not studied (Kukulski et al, 2016). Finally, in S. pombe , the AP-2 α component (Apl3p) was shown to interact physically with clathrin light chain (Clc1p), and genetically with both Clc1p and the endocytic factor Sla2p and additionally apl3Δ null mutants showed altered dynamics of endocytic sites associated with abnormal cell wall synthesis and morphogenesis (de León et al, 2016; de León and Valdivieso, 2016). …”

Papel de los adaptadores de clatrina GGA y Ent3, y de la SNARE Fsv1 en el tráfico entre la red trans del Golgi, el endosoma prevacuolar y la vacuola

“…En S. cerevisiae se ha visto interacción física de la clatrina con el adaptador AP-1 pero no con los otros dos adaptadores (Lemmon and Traub, 2000;Yeung et al, 1999). En S. pombe por el momento sólo se ha detectado interacción física de AP-2 con clatrina (de Leon et al, 2016).…”