CIE is thought to be a nonselective process for internalization of bulk membrane (Hansen and Nichols, 2009
; Sandvig et al., 2011
). Here we show that after endocytosis of bulk membrane by CIE, some cargo proteins (CD44, CD98, and CD147) are recognized and sorted on endosomes by Hook1, acting with Rab22a, to facilitate rapid routing of this cargo to recycling endosomes, avoiding the default route to EEA1-associated endosomes and lysosomes.
An examination of the cytoplasmic tails of all CIE cargo proteins reveals no common sequence information, which is consistent with the lack of cargo selection at the endocytic step. However, an altered intracellular itinerary is taken by some of these cargo proteins (CD147, CD44, and CD98; Eyster et al., 2009
), and in this study we identified cytoplasmic sequences in CD147 and CD44 that were sufficient to redirect the trafficking of Tac, a reporter protein. One feature common to the cytoplasmic tails of CD147, CD44, and CD98 is the presence of acidic amino acid pairs or clusters. In contrast, the cargo that traffics along the default pathway (Tac, MHCI, Glut1, and GPI-APs CD55 and CD59) lacks such di-acidic residues. The acidic residues in CD147 contributed to endosomal sorting in that mutation of these residues led to impaired sorting and lack of interaction in the Y2H screen. However, sorting was not completely lost when the acidic residues were mutated, which suggests that other sequences may contribute to the sorting. A sorting role for a single leucine (at position 248 in the human sequence) in basolateral targeting of CD147 in MDCK cells has been reported (Deora et al., 2004
); however, we did not observe altered trafficking of a leucine mutant of CD147 (unpublished data). Acidic clusters have been observed in other PM proteins entering cells by CIE. The inwardly rectifying K channel, Kir3.4, contains clusters of acidic amino acids that were found to be critical for maintaining these channels at the cell surface (Gong et al., 2007
). As these proteins become trapped in vacuoles generated by expression of Arf6 Q67L, it is likely that Kir3.4 travels along the CIE pathway, is sorted along with CD147, CD44, and CD98, and might be sorted via Hook1.
The evidence for sorting of proteins on endosomes is increasing and has been observed for those proteins entering by CIE and CME. Distinct cytoplasmic sequences on G protein–coupled receptors promote sorting out of early endosomes to facilitate receptor recycling after ligand-stimulated CME (Puthenveedu et al., 2010
). Phenylalanine-containing clusters in the cytoplasmic portion of the TfR may be important for normal rate of recycling of the receptor (Dai et al., 2004
). Interestingly, two cargo proteins that enter cells by the CIE pathway described here, CD44 and syndecan 2, both contain carboxyl-terminal PDZ binding motifs. In the case of syndecan 2, the PDZ domain protein syntenin is required for syndecan 2 to recycle back to the surface (Zimmermann et al., 2005
). The identification of the machinery that recognizes and sorts these different cargo proteins will likely include roles for sorting nexins, ubiquitin-interacting proteins, and the known Rabs and EHD proteins implicated in various recycling pathways (Grant and Donaldson, 2009
; Naslavsky and Caplan, 2011
We found that Hook1 specifically binds to CD147 and facilitates the microtubule-dependent sorting of CD147, CD44, and CD98 away from EEA1-positive endosomes and into recycling tubules. Hook1 is well suited to perform such a function, as it contains an amino-terminal domain associated with microtubule binding and a carboxyl-terminal domain associated with cargo binding. The Hook protein was originally identified in Drosophila melanogaster
, where it functions in the trafficking to late endosomes (Krämer and Phistry, 1996
; Sunio et al., 1999
). There are three mammalian Hook proteins. Hook2 has been localized to the centrosome (Szebenyi et al., 2007
) and Golgi (Baron Gaillard et al., 2011
), and has been shown to be involved in the formation of the primary cilium (Baron Gaillard et al., 2011
). Hook3 also localizes to the Golgi (Walenta et al., 2001
). We found partial colocalization of endogenous Hook1 with CD147 and other CIE cargo proteins in tubular endosomes, and this localization was enhanced in the absence of microtubules. We could detect a biochemical interaction of CD147 and CD98 with the carboxyl-terminal domain of Hook1, but not with the full-length protein. This might be caused by some type of autoinhibitory or regulatory interaction of the full-length protein that we are currently examining in more detail. Two other proteins have been identified that interact with the carboxyl-terminal region of Hook1. These are the Vps18 subunit of the HOPs complex (Richardson et al., 2004
) and AKTIP (Xu et al., 2008
), an E2 ubiquitin-conjugating enzyme also known as FTS. We do not know whether the interaction of these proteins with Hook1 plays a role in sorting of CD147, but the fact that these proteins may form larger complexes suggests that the Hook1-associated machinery interacting with and sorting CIE cargo proteins might involve other components.
Indeed, another component of the sorting machinery is Rab22a, which we previously showed to be required for the formation of the recycling endosomal tubules in HeLa cells (Weigert et al., 2004
). Here we extend Rab22a function to that of assisting Hook1 in the sorting of CIE cargo (CD44, CD98, and CD147) into the tubules. Hook1 has been shown to coimmunoprecipitate with overexpressed Rab7, Rab9, and Rab11, which suggests that Hook1 might interact with endosomal Rabs (Luiro et al., 2004
); however, for Rab22a, we could not see a direct interaction with Hook1. Although we could not detect direct biochemical interaction between Hook1 and Rab22a, these two proteins seem to be acting at the same sorting step in cells, given that each effectively rescued the dominant-negative phenotypes induced by the other. Because dominant-negative proteins often sequester essential components, this striking characteristic for Hook1 and Rab22 suggests that they may be interacting with additional components to relieve the mutant phenotypes. Rab22a has been implicated in TfR recycling in other cells (Magadán et al., 2006
) and in preventing phagosome maturation in cells harboring mycobacteria (Roberts et al., 2006
), a phenotype of lysosome avoidance that is consistent with observations reported here. A similar coordination between a Hook and a Rab has been observed in that Rab8 overexpression suppresses the effect of Hook2 knockdown on ciliogenesis (Baron Gaillard et al., 2011
). Given the microtubule dependence of cargo sorting and the connection of Hook1 with microtubules, it is likely that Hook1 and Rab22a act together with a microtubule motor protein such as a kinesin or dynein (Horgan and McCaffrey, 2011
This sorting at the level of the endosome provides a means to monitor protein quality control of cell surface proteins. By sampling the PM by bulk endocytosis, PM proteins can be monitored and sorted, allowing some proteins to recycle back out to the PM, and for misfolded or malfunctioning, ubiquitinated cargo to be sent on to degradation. Further investigations will identify additional signals that can influence this sorting; e.g., how ubiquitination of CIE cargo proteins could affect their sorting on endosomes.
The proteins that are directly sorted into recycling tubules, CD44, CD98, and CD147, are all implicated in cell–cell and cell–matrix interactions. As such, they may be important cell surface components that influence cell adhesion and migratory properties, and as we observed here are important for cell spreading. CD44 is a hyaluronan receptor (Zöller, 2011
) and can influence growth factor signaling (Ponta et al., 2003
). CD98 plays an important role in nutrient uptake through its association with amino acid transporters (Yan et al., 2008
), and interacts with and influences integrin trafficking and signaling (Cantor and Ginsberg, 2012
). CD147, also known as Basigin or EMMPRIN (extracellular matrix metalloproteinase inducer) associates with integrins and monocarboxylate transporters, and interacts with matrix metalloproteases (Iacono et al., 2007
). Levels of CD44, CD98, and CD147 are elevated in many cancers, which suggests that the rapid recycling and avoidance of degradation observed here could contribute to this phenotype. The ability of cells to segregate and rapidly recycle these proteins might be carefully regulated in tissues during development and turned on during wound healing and cancer cell metastasis. Identifying additional sorting components and understanding the mechanism of how these proteins are sorted on endosomes could lead to novel therapeutic targets.