The actin-binding protein Hip1R associates with clathrin during early stages of endocytosis and promotes clathrin assembly in vitro.
Huntingtin-interacting protein 1 related (Hip1R) is a novel component of clathrin-coated pits and vesicles and is a mammalian homologue of Sla2p, an actin-binding protein important for both actin organization and endocytosis in yeast. Here, we demonstrate that Hip1R binds via its putative central coiled-coil domain to clathrin, and provide evidence that Hip1R and clathrin are associated in vivo at sites of endocytosis. First, real-time analysis of Hip1R-YFP and DsRed-clathrin light chain (LC) in live cells revealed that these proteins show almost identical temporal and spatial regulation at the cell cortex. Second, at the ultrastructure level, immunogold labeling of 'unroofed' cells showed that Hip1R localizes to clathrin-coated pits. Third, overexpression of Hip1R affected the subcellular distribution of clathrin LC. Consistent with a functional role for Hip1R in endocytosis, we also demonstrated that it promotes clathrin cage assembly in vitro. Finally, we showed that Hip1R is a rod-shaped apparent dimer with globular heads at either end, and that it can assemble clathrin-coated vesicles and F-actin into higher order structures. In total, Hip1R's properties suggest an early endocytic function at the interface between clathrin, F-actin, and lipids.
Engqvist-Goldstein
A E
AE
Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA 94720, USA.
Warren
R A
RA
Kessels
M M
MM
Keen
J H
JH
Heuser
J
J
Drubin
D G
DG
D000199:Actins / Q000378:metabolism; D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology*; D002966:Clathrin / Q000378:metabolism* / Q000502:physiology; D022163:Clathrin-Coated Vesicles / Q000737:chemistry / Q000187:drug effects / Q000648:ultrastructure; D018389:Codon, Nonsense; D004268:DNA-Binding Proteins; D019281:Dimerization; D004705:Endocytosis / Q000502:physiology*; D006651:Histocytochemistry; D021621:Imaging, Three-Dimensional; D051379:Mice; D008840:Microfilament Proteins; D008854:Microscopy, Electron; D018715:Microscopy, Video; D011233:Precipitin Tests; D011485:Protein Binding; D013347:Subcellular Fractions / Q000737:chemistry; D013997:Time Factors
D000199:Actins; D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D002966:Clathrin; D018389:Codon, Nonsense; D004268:DNA-Binding Proteins; C489536:Hip1 protein, mouse; C489534:Hip1r protein, mouse; D008840:Microfilament Proteins