An actin-binding protein of the Sla2/Huntingtin interacting protein 1 family is a novel component of clathrin-coated pits and vesicles

Abstract

The actin cytoskeleton has been implicated in endocytosis, yet few molecules that link these systems have been identified. Here, we have cloned and characterized mHip1R, a protein that is closely related to huntingtin interacting protein 1 (Hip1). These two proteins are mammalian homologues of Sla2p, an actin binding protein important for actin organization and endocytosis in yeast. Sequence alignments and secondary structure predictions verified that mHip1R belongs to the Sla2 protein family. Thus, mHip1R contains an NH(2)-terminal domain homologous to that implicated in Sla2p's endocytic function, three predicted coiled-coils, a leucine zipper, and a talin-like actin-binding domain at the COOH terminus. The talin-like domain of mHip1R binds to F-actin in vitro and colocalizes with F-actin in vivo, indicating that this activity has been conserved from yeast to mammals. mHip1R shows a punctate immunolocalization and is enriched at the cell cortex and in the perinuclear region. We concluded that the cortical localization represents endocytic compartments, because mHip1R colocalizes with clathrin, AP-2, and endocytosed transferrin, and because mHip1R fractionates biochemically with clathrin-coated vesicles. Time-lapse video microscopy of mHip1R-green fluorescence protein (GFP) revealed a blinking behavior similar to that reported for GFP-clathrin, and an actin-dependent inward movement of punctate structures from the cell periphery. These data show that mHip1R is a component of clathrin-coated pits and vesicles and suggest that it might link the endocytic machinery to the actin cytoskeleton.

Figure 1

Analysis of mHip1R sequence. (a) Comparison of the full-length aa sequence identities of Sla2-related proteins. The percentage identity for the two most similar proteins is highlighted in bold. For hHip1R, the cDNA sequence reported by Ishikawa et al. 1998 was used (KIAA0655 protein; sequence data available from EMBL/GenBank/DDBJ under accession no. AB014555). (b) Sequence alignment of the NH₂-terminal domain in Sla2-related proteins. Residues that are conserved in at least four proteins are boxed. For hHip1R, residue M179, designated as the first methionine by Seki et al. 1998, is highlighted in bold. The upstream hHip1R sequence is from Ishikawa et al. 1998. The hHip1 sequence displayed represents a sequence published by Kalchman et al. 1997 (M82-A277), and a recently identified upstream sequence (S1-D81) (Chopra, V.S., unpublished results). (c) Schematic diagram showing mHip1R domain organization. The three predicted coiled–coil forming domains and the leucine zipper are indicated, as well as the COOH-terminal talin-like domain, and the NH₂-terminal domain. The Coils program (Lupas et al. 1991) was used to determine the probability of coiled–coil formation.

Figure 2

Mouse mHip1R mRNA and protein expression levels. (a) mHip1R mRNA expression examined by Northern blotting during the course of mouse embryonic development. The numbers at the top of each lane represent days after fertilization. (b) mHip1R mRNA expression in different tissues of adult mice. The Northern probe comprised 297 bp at the COOH terminus corresponding to aa 970–1068. (c and d) Actin expression is shown as a control for RNA integrity and loading. (e) mHip1R protein expression examined by Western blotting in different cell lines. Approximately 50 μg protein from postnuclear tissue homogenates was loaded per lane. (f) mHip1R protein expression in different adult mouse tissues. Approximately 200 μg of protein was loaded per lane.

Figure 3

The talin-like domain of mHip1R binds to F-actin. (a) SDS-PAGE of a cosedimentation assay. The gels were stained with Coomassie blue, where P represents the pellets and S represents the supernatants. 0–10 μM nonmuscle actin was used with a constant concentration of GST-talin domain (1.5 μM). (b) Percentage of GST-talin domain bound to actin as a function of actin concentration. GST alone was used as a control. This experiment was repeated three times with similar results.

Figure 4

Indirect immunofluorescence of endogenous Hip1R in COS-7 cells. (a, d, and g) Labeling of Hip1R by GP#8 and anti–guinea pig FITC secondary antibody. (b, e, and h) F-actin staining labeled by rhodamine-phalloidin. (c, f, and i) Overlay between Hip1R and F-actin. In c this merge also includes 4,6-diamidino-2-phenylindole dihydrochloride staining to label the nucleus. Bars, 10 μM. (a–c) Punctate vesicle-like Hip1R localization that is enriched in the perinuclear area (a, arrow). (d–f) Hip1R-positive puncta are enriched at the actin-rich cell cortex as demonstrated by confocal microscopy sectioning through a medial plane of a clump of cells. (g–i and enlargements) Hip1R is present in some F-actin–rich ruffles, where the staining is less punctate and more continuous and colocalizes partially with F-actin. Confocal images close to the cell attachment surface.

Figure 5

mHip1R colocalizes with clathrin and AP-2 in COS-7 and NIH/3T3 fibroblasts. (a, d, g, and j) mHip1R. (b, e, and h) Clathrin. (k) AP-2. (c, f, i, and l) Overlay. (a–c) COS-7 cells. mHip1R and clathrin show a similar subcellular distribution, including enrichments in the perinuclear areas. Bar, 10 μM. (d–f and insets) Confocal microscopy of COS-7 cells. mHip1R largely colocalizes with clathrin in the perinuclear region. Bar, 5 μM. (g–i and enlargements) NIH/3T3 cells. mHip1R partially colocalizes with clathrin at the cell cortex. Bar, 10 μM. (j–l) COS-7 cells. Colocalization of mHip1R and AP-2. Bar, 10 μM.

Figure 6

Hip1R partially colocalizes with transferrin-labeled endocytic compartment in COS-7 cells. (a and d) mHip1R. (b and e) Transferrin–Texas red. (c and f) Overlay. (a–c) At 2 min of uptake, transferrin labels the cell cortex (confocal medial section of cell clump). Hip1R-positive puncta overlaps with this cortical staining. (d–f and enlargements) At 10 min of transferrin uptake, Hip1R partially colocalizes with transferrin-labeled endocytic compartment. The confocal images are showing a section close to the attachment surface as observed by confocal microscopy. Bars, 10 μM.

Figure 7

mHip1R is a peripheral membrane protein that cofractionates with clathrin-coated vesicles. (a) Subcellular fractionation of mHip1R. Different fractions of mouse brain homogenate were resolved by SDS-PAGE and examined for the indicated proteins by immunoblotting. The P2 fractions were extracted under different conditions as indicated in the figure. For each pellet (P) and supernatant (S) fraction, equal amounts of protein were loaded. (b and c) Purification of clathrin-coated vesicles from mouse brain homogenate. Fraction (V) represents enriched clathrin-coated vesicles. Equivalent portions of S1, P2, and S2 were loaded (∼0.02% of the fraction), and equivalent portions of S3, P3, S4, and V were loaded (∼2% of the fraction). (b) Coomassie-stained gel of the fractions. Arrow indicates the band corresponding to the clathrin–heavy chain enriched in the vesicle fraction. (c) Immunoblotting of fractions.

Figure 7

mHip1R is a peripheral membrane protein that cofractionates with clathrin-coated vesicles. (a) Subcellular fractionation of mHip1R. Different fractions of mouse brain homogenate were resolved by SDS-PAGE and examined for the indicated proteins by immunoblotting. The P2 fractions were extracted under different conditions as indicated in the figure. For each pellet (P) and supernatant (S) fraction, equal amounts of protein were loaded. (b and c) Purification of clathrin-coated vesicles from mouse brain homogenate. Fraction (V) represents enriched clathrin-coated vesicles. Equivalent portions of S1, P2, and S2 were loaded (∼0.02% of the fraction), and equivalent portions of S3, P3, S4, and V were loaded (∼2% of the fraction). (b) Coomassie-stained gel of the fractions. Arrow indicates the band corresponding to the clathrin–heavy chain enriched in the vesicle fraction. (c) Immunoblotting of fractions.

Figure 8

Expression of mHip1R domains in COS-7 cells reveals that mHip1R contains at least two different localization signals. Full-length and different domains of mHip1R were expressed as GFP fusion proteins and/or myc-tagged proteins. (a) Schematic diagram of mHip1R domains expressed. (b) Immunoblotting of extracts from cells expressing the different mHip1R constructs. GFP fusion proteins were detected with anti-GFP antibodies. myc-tagged proteins were detected with anti-myc antibodies. (c–h) The upper panels show subcellular localization of the constructs. The lower panels show rhodamine-phalloidin staining of F-actin in the same cells. (c) mHip1R (aa 1–1068)–GFP. (d) mHip1R (aa 1–1068)–6 myc. (e) mHip1R (aa 766–1068)–GFP. (f) mHip1R (aa 346–1068)–GFP. (g) mHip1R (aa 1–324)–6 myc. (h) mHip1R (aa 1–655)–6 myc. Bars, 10 μm.

Figure 8

Expression of mHip1R domains in COS-7 cells reveals that mHip1R contains at least two different localization signals. Full-length and different domains of mHip1R were expressed as GFP fusion proteins and/or myc-tagged proteins. (a) Schematic diagram of mHip1R domains expressed. (b) Immunoblotting of extracts from cells expressing the different mHip1R constructs. GFP fusion proteins were detected with anti-GFP antibodies. myc-tagged proteins were detected with anti-myc antibodies. (c–h) The upper panels show subcellular localization of the constructs. The lower panels show rhodamine-phalloidin staining of F-actin in the same cells. (c) mHip1R (aa 1–1068)–GFP. (d) mHip1R (aa 1–1068)–6 myc. (e) mHip1R (aa 766–1068)–GFP. (f) mHip1R (aa 346–1068)–GFP. (g) mHip1R (aa 1–324)–6 myc. (h) mHip1R (aa 1–655)–6 myc. Bars, 10 μm.

Figure 8

Expression of mHip1R domains in COS-7 cells reveals that mHip1R contains at least two different localization signals. Full-length and different domains of mHip1R were expressed as GFP fusion proteins and/or myc-tagged proteins. (a) Schematic diagram of mHip1R domains expressed. (b) Immunoblotting of extracts from cells expressing the different mHip1R constructs. GFP fusion proteins were detected with anti-GFP antibodies. myc-tagged proteins were detected with anti-myc antibodies. (c–h) The upper panels show subcellular localization of the constructs. The lower panels show rhodamine-phalloidin staining of F-actin in the same cells. (c) mHip1R (aa 1–1068)–GFP. (d) mHip1R (aa 1–1068)–6 myc. (e) mHip1R (aa 766–1068)–GFP. (f) mHip1R (aa 346–1068)–GFP. (g) mHip1R (aa 1–324)–6 myc. (h) mHip1R (aa 1–655)–6 myc. Bars, 10 μm.

Figure 9

Time-lapse video microscopy of mHip1R–GFP in COS-7 cells. (a and b) The distribution of mHip1R–GFP in a cell before (a) and 45 s after (b) addition of Triton X-100 shows that the cortical staining is retained in extracted cells. Bar, 10 μm. (c) Images at 1.5-s intervals show that mHip1R–GFP puncta exhibit dynamic behavior. Changes in individual mHip1R–GFP puncta throughout this series of images are summarized in the diagram to the right, in which persistent (filled circles), disappearing (open circles), and appearing (asterisks) puncta are indicated. Bar, 2.5 μm.

Figure 10

Time-lapse video microscopy of mHip1R–GFP in NIH/3T3 fibroblasts. (a–c) The first image in each series shows a low magnification overview of the cell. Bars in the figures serve as stationary reference points. (a) Images taken at 20-s intervals show movement of mHip1R–GFP away from the cell periphery (see arrows and arrowheads). Puncta further away from the cell periphery often appear to align on linear tracks (see arrows). Bar, 10 μm. (b) Cell before treatment with LAT-A. Puncta are moving inwards from the cell periphery (see arrows and arrowheads). (c) Same cell 5 min after adding LAT-A. The puncta no longer show a net movement away from the cell periphery. Bar, 10 μm.