Dynamic early recruitment of GAK-Hsc70 regulates coated pit maturation

Clathrin-mediated endocytosis (CME) is the process by which clathrin assembles on the plasma membrane to form clathrin-coated pits (CCPs), which then invaginate, accumulate cargo and are released by fission from the membrane to form clathrin-coated vesicles (CCVs). A transition of nascent CCPs from flat-to-curved has been observed by various methods. However, what drives this transition remains unknown and controversial. GAK and its chaperone protein, Hsc70, are well-known to mediate clathrin release from CCVs and several studies have observed a late burst of GAK recruitment as CCVs form. Other studies have proposed that early recruitment of GAK-Hsc70 could function to provide the necessary energy source to remodel nascent flat clathrin lattices, replacing hexagons with pentagons and enabling a gain of curvature and invagination of the growing CCP; however, direct functional evidence is lacking. Here we show that GAK knockdown inhibits CCP formation and invagination. Furthermore, mutations in the J domain of GAK that abolish Hsc70 recruitment to and activation at CCPs, lead to the accumulation of GAK at CCPs, hinder CCP stabilization and invagination and result in a striking increase in the proportion of short-lived, abortive CCPs. These findings support the hypothesis that GAK-Hsc70 promotes the turnover and remodeling of nascent clathrin assemblies required for curvature development during CME. Significance StatementGAK and its chaperone protein, Hsc70, are known to be recruited to clathrin-coated vesicles (CCVs) to mediate clathrin uncoating. Previous studies have proposed that early recruitment of GAK-Hsc70 to CCPs could function to remodel nascent flat clathrin lattices, replacing hexagons with pentagons and enabling a gain of curvature of the assembling polymeric coat. However, there are conflicting views, and direct functional evidence is lacking. Here we show that GAK knockdown inhibits CCP formation and invagination. A detailed domain-specific mutational analysis of GAK pinpointed the importance of J domain-Hsc70 interactions in regulating crucial early steps of CME including CCP stabilization and invagination. These findings support a hypothesis that GAK-Hsc70 promotes turnover of clathrin at nascent CCPs required for curvature development.