Clathrin Hub Expression Affects Early Endosome Distribution with Minimal Impact on Receptor Sorting and Recycling

Abstract

Clathrin-coated vesicles execute receptor-mediated endocytosis at the plasma membrane. However, a role for clathrin in later endocytic trafficking processes, such as receptor sorting and recycling or maintaining the organization of the endocytic pathway, has not been thoroughly characterized. The existence of clathrin-coated buds on endosomes suggests that clathrin might mediate later endocytic trafficking events. To investigate the function of clathrin-coated buds on endosomal membranes, endosome function and distribution were analyzed in a HeLa cell line that expresses the dominant-negative clathrin inhibitor Hub in an inducible manner. As expected, Hub expression reduced receptor-mediated endocytosis at the plasma membrane. Hub expression also induced a perinuclear aggregation of early endosome antigen 1-positive early endosomes, such that sorting and recycling endosomes were found tightly concentrated in the perinuclear region. Despite the dramatic redistribution of endosomes, Hub expression did not affect the overall kinetics of receptor sorting or recycling. These data show that clathrin function is necessary to maintain proper cellular distribution of early endosomes but does not play a prominent role in sorting and recycling events. Thus, clathrin's role on endosomal membranes is to influence organelle localization and is distinct from its role in trafficking pathways at the plasma membrane and trans-Golgi network. INTRODUCTIONClathrin-coated vesicles (CCV) mediate the selective transport of integral membrane proteins between some cellular membranes (Kirchhausen, 2000). At the plasma membrane, CCV facilitate receptor-mediated endocytosis (RME), whereby cell surface receptors and their ligands are internalized and delivered to the endocytic pathway (Schwartz, 1995). After RME, the fates of internalized receptors and ligands vary (reviewed by Mukherjee et al., 1997). Some progress to the late endocytic pathway where they are degraded in lysosomes (Carpenter and Cohen, 1976;Fox and Das, 1979). Others, such as transferrin (Tf) and low density lipoprotein (LDL) receptors, are sorted to a recycling compartment (Dunn et al., 1989;Mayor et al., 1993), from which they return to the plasma membrane (Anderson et al., 1982;Hopkins and Trowbridge, 1983). Although it is known that these receptors are endocytosed via CCV (Anderson et al., 1977;Willingham et al., 1979;Bleil and Bretscher, 1982), the degree to which clathrin mediates subsequent sorting and recycling of the proteins has been a subject of debate.The early endocytic pathway is composed of both sorting and recycling endosomes (Ghosh et al., 1994). Sorting endosomes separate endocytosed material that is to be recycled from material destined for the lysosome. Recycling components proceed to recycling endosomes from which they return to the plasma membrane. Clathrin-coated buds have been observed on early endosomes (Stoorvogel et al., 1996), making it a logical hypothesis that clathrin might mediate receptor sorting in the recycling pathway. A compelling findin...

The adaptor complex AP-2 regulates post-endocytic trafficking through the non-clathrin Arf6-dependent endocytic pathway

“…1C, arrows). While such a phenotype has not previously been described in clathrin-depleted cells, it has been reported in cells overexpressing the clathrin hub region (Bennett et al, 2001), which functions in a dominant-negative manner. Similar Tfn localization has also been reported in cells expressing a dynamin mutant that blocks CME (van Dam and Stoorvogel, 2002), a phenotype that was also observed in our system (Fig.…”

In Vitro Formation of Recycling Vesicles from Endosomes Requires Adaptor Protein-1/Clathrin and Is Regulated by Rab4 and the Connector Rabaptin-5

“…These results thus support the earlier observations that transferrin receptor recycling in living cells at least in part was affected by BFA and involved dynamin . Perturbation of clathrin function in living cells was always found to affect endocytosis more effectively than receptor recycling (Bennett et al, 2001;Wettey et al, 2002;Moskowitz et al, 2003). The reduced recycling rates thus could be interpreted as an indirect result of disturbing clathrin-dependent pathways from the plasma membrane, and from and to the TGN, rather than the result of a direct involvement of clathrin in recycling.…”

“…Perturbation of clathrin coat formation by regulated expression of a dominant-negative clathrin hub domain in HeLa cells reduced endocytosis by ϳ50% and caused perinuclear aggregation of early endosomes, but it reduced recycling by only ϳ20% (Bennett et al, 2001). Similarly, drug-induced cross-linking of an FK506-binding protein-clathrin light-chain fusion, which dominantly interferes with clathrin function, reduced LDL or transferrin uptake by half, but minimally affected receptor recycling in Chinese hamster ovary (CHO) cells (Moskowitz et al, 2003).…”

Clathrin Terminal Domain-Ligand Interactions Regulate Sorting of Mannose 6-Phosphate Receptors Mediated by AP-1 and GGA Adaptors

“…Furthermore, so-called gyrating clathrin (G-clathrin) has been suggested to operate in an Arf6-dependent rapid recycling route (27). By contrast, dominant interference with clathrin function by inducible overexpression of the clathrin hub domain or ligand-induced cross-linking of clathrin LCs had only minor effects on transferrin recycling (28,29). Likewise, it remains unclear to what degree flat clathrin coats on endosomes are required for Hrs-mediated degradative sorting of epidermal growth factor (23).…”