A role for EHD4 in the regulation of early endosomal transport.
All four of the C-terminal Eps15 homology domain (EHD) proteins have been implicated in the regulation of endocytic trafficking. However, the high level of amino acid sequence identity among these proteins has made it challenging to elucidate the precise function of individual EHD proteins. We demonstrate here with specific peptide antibodies that endogenous EHD4 localizes to Rab5-, early embryonic antigen 1 (EEA1)- and Arf6-containing endosomes and colocalizes with internalized transferrin in the cell periphery. Knock-down of EHD4 expression by both small interfering RNA and short hairpin RNA leads to the generation of enlarged early endosomal structures that contain Rab5 and EEA1 as well as internalized transferrin or major histocompatibility complex class I molecules. In addition, cargo destined for degradation, such as internalized low-density lipoprotein, also accumulates in the enlarged early endosomes in EHD4-depleted cells. Moreover, we have demonstrated that these enlarged early endosomes are enriched in levels of the activated GTP-bound Rab5. Finally, we show that endogenous EHD4 and EHD1 interact in cells, suggesting coordinated involvement in the regulation of receptor transport along the early endosome to endocytic recycling compartment axis. The results presented herein provide evidence that EHD4 is involved in the control of trafficking at the early endosome and regulates exit of cargo toward both the recycling compartment and the late endocytic pathway.
Sharma
Mahak
M
Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE 68198-5870, USA.
Naslavsky
Naava
N
Caplan
Steve
S
D001692:Biological Transport; D004268:DNA-Binding Proteins / Q000378:metabolism*; D011992:Endosomes / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism*; D011485:Protein Binding; D021381:Protein Transport; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000494:pharmacology; D013997:Time Factors; D033921:Vesicular Transport Proteins / Q000378:metabolism; D020696:rab5 GTP-Binding Proteins / Q000378:metabolism
D004268:DNA-Binding Proteins; C120700:EHD1 protein, human; C406448:EHD4 protein, human; D009687:Nuclear Proteins; D034741:RNA, Small Interfering; D033921:Vesicular Transport Proteins; D020696:rab5 GTP-Binding Proteins