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1.
Legendre-Guillemin V Metzler M Charbonneau M Gan L Chopra V Philie J Hayden MR McPherson PS 《The Journal of biological chemistry》2002,277(22):19897-19904
Huntingtin-interacting protein 1 (HIP1) and HIP12 are orthologues of Sla2p, a yeast protein with essential functions in endocytosis and regulation of the actin cytoskeleton. We now report that HIP1 and HIP12 are major components of the clathrin coat that interact but differ in their ability to bind clathrin and the clathrin adaptor AP2. HIP1 contains a clathrin-box and AP2 consensus-binding sites that display high affinity binding to the terminal domain of the clathrin heavy chain and the ear domain of the AP2 alpha subunit, respectively. These consensus sites are poorly conserved in HIP12 and correspondingly, HIP12 does not bind to AP2 nor does it demonstrate high affinity clathrin binding. Moreover, HIP12 co-sediments with F-actin in contrast to HIP1, which exhibits no interaction with actin in vitro. Despite these differences, both proteins efficiently stimulate clathrin assembly through their central helical domain. Interestingly, in both HIP1 and HIP12, this domain binds directly to the clathrin light chain. Our data suggest that HIP1 and HIP12 play related yet distinct functional roles in clathrin-mediated endocytosis. 相似文献
2.
Scd5p and clathrin function are important for cortical actin organization,endocytosis, and localization of sla2p in yeast
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Henry KR D'Hondt K Chang J Newpher T Huang K Hudson RT Riezman H Lemmon SK 《Molecular biology of the cell》2002,13(8):2607-2625
SCD5 was identified as a multicopy suppressor of clathrin HC-deficient yeast. SCD5 is essential, but an scd5-Delta338 mutant, expressing Scd5p with a C-terminal truncation of 338 amino acids, is temperature sensitive for growth. Further studies here demonstrate that scd5-Delta338 affects receptor-mediated and fluid-phase endocytosis and normal actin organization. The scd5-Delta338 mutant contains larger and depolarized cortical actin patches and a prevalence of G-actin bars. scd5-Delta338 also displays synthetic negative genetic interactions with mutations in several other proteins important for cortical actin organization and endocytosis. Moreover, Scd5p colocalizes with cortical actin. Analysis has revealed that clathrin-deficient yeast also have a major defect in cortical actin organization and accumulate G-actin. Overexpression of SCD5 partially suppresses the actin defect of clathrin mutants, whereas combining scd5-Delta338 with a clathrin mutation exacerbates the actin and endocytic phenotypes. Both Scd5p and yeast clathrin physically associate with Sla2p, a homologue of the mammalian huntingtin interacting protein HIP1 and the related HIP1R. Furthermore, Sla2p localization at the cell cortex is dependent on Scd5p and clathrin function. Therefore, Scd5p and clathrin are important for actin organization and endocytosis, and Sla2p may provide a critical link between clathrin and the actin cytoskeleton in yeast, similar to HIP1(R) in animal cells. 相似文献
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Brett TJ Legendre-Guillemin V McPherson PS Fremont DH 《Nature structural & molecular biology》2006,13(2):121-130
Huntingtin-interacting protein-1 related (HIP1R) has a crucial protein-trafficking role, mediating associations between actin and clathrin-coated structures at the plasma membrane and trans-Golgi network. Here, we characterize the F-actin-binding region of HIP1R, termed the talin-HIP1/R/Sla2p actin-tethering C-terminal homology (THATCH) domain. The 1.9-A crystal structure of the human HIP1R THATCH core reveals a large sequence-conserved surface patch created primarily by residues from the third and fourth helices of a unique five-helix bundle. Point mutations of seven contiguous patch residues produced significant decreases in F-actin binding. We also show that THATCH domains have a conserved C-terminal latch capable of oligomerizing the core, thereby modulating F-actin engagement. Collectively, these results establish a framework for investigating the links between endocytosis and actin dynamics mediated by THATCH domain-containing proteins. 相似文献
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Huntington's disease is a genetic neurological disorder that is triggered by the dissociation of the huntingtin protein (htt) from its obligate interaction partner Huntingtin-interacting protein 1 (HIP1). The release of the huntingtin protein permits HIP1 protein interactor (HIPPI) to bind to its recognition site on HIP1 to form a HIPPI/HIP1 complex that recruits procaspase-8 to begin the process of apoptosis. The interaction module between HIPPI and HIP1 was predicted to resemble a death-effector domain. Our 2.8-Å crystal structure of the HIP1 371-481 subfragment that includes F432 and K474, which is important for HIPPI binding, is not a death-effector domain but is a partially opened coiled coil. The HIP1 371-481 model reveals a basic surface that we hypothesize to be suitable for binding HIPPI. There is an opened region next to the putative HIPPI site that is highly negatively charged. The acidic residues in this region are highly conserved in HIP1 and a related protein, HIP1R, from different organisms but are not conserved in the yeast homologue of HIP1, sla2p. We have modeled ∼ 85% of the coiled-coil domain by joining our new HIP1 371-481 structure to the HIP1 482-586 model (Protein Data Bank code: 2NO2). Finally, the middle of this coiled-coil domain may be intrinsically flexible and suggests a new interaction model where HIPPI binds to a U-shaped HIP1 molecule. 相似文献
7.
Metzler M Legendre-Guillemin V Gan L Chopra V Kwok A McPherson PS Hayden MR 《The Journal of biological chemistry》2001,276(42):39271-39276
Polyglutamine expansion in huntingtin is the underlying mutation leading to neurodegeneration in Huntington disease. This mutation influences the interaction of huntingtin with different proteins, including huntingtin-interacting protein 1 (HIP1), in which affinity to bind to mutant huntingtin is profoundly reduced. Here we demonstrate that HIP1 colocalizes with markers of clathrin-mediated endocytosis in neuronal cells and is highly enriched on clathrin-coated vesicles (CCVs) purified from brain homogenates. HIP1 binds to the clathrin adaptor protein 2 (AP2) and the terminal domain of the clathrin heavy chain, predominantly through a small fragment encompassing amino acids 276-335. This region, which contains consensus clathrin- and AP2-binding sites, functions in conjunction with the coiled-coil domain to target HIP1 to CCVs. Expression of various HIP1 fragments leads to a potent block of clathrin-mediated endocytosis. Our findings demonstrate that HIP1 is a novel component of the endocytic machinery. 相似文献
8.
Mishra SK Agostinelli NR Brett TJ Mizukami I Ross TS Traub LM 《The Journal of biological chemistry》2001,276(49):46230-46236
Clathrin-mediated endocytosis is a major pathway for the internalization of macromolecules into the cytoplasm of eukaryotic cells. The principle coat components, clathrin and the AP-2 adaptor complex, assemble a polyhedral lattice at plasma membrane bud sites with the aid of several endocytic accessory proteins. Here, we show that huntingtin-interacting protein 1 (HIP1), a binding partner of huntingtin, copurifies with brain clathrin-coated vesicles and associates directly with both AP-2 and clathrin. The discrete interaction sequences within HIP1 that facilitate binding are analogous to motifs present in other accessory proteins, including AP180, amphiphysin, and epsin. Bound to a phosphoinositide-containing membrane surface via an epsin N-terminal homology (ENTH) domain, HIP1 associates with AP-2 to provide coincident clathrin-binding sites that together efficiently recruit clathrin to the bilayer. Our data implicate HIP1 in endocytosis, and the similar modular architecture and function of HIP1, epsin, and AP180 suggest a common role in lipid-regulated clathrin lattice biogenesis. 相似文献
9.
Caruso JA Reiners JJ 《Apoptosis : an international journal on programmed cell death》2006,11(11):1877-1885
BID is an essential component of many apoptotic pathways. Cytosolic proteases cleave BID within an extended loop region, generating
an active truncated fragment which synergizes with BAX and BAK to induce release of apoptogenic factors from mitochondria.
To determine whether other proteins are cleaved in a similar manner as BID, we performed a database search for proteins which
possess sequence similarity with the BID loop region. One of the proteins identified was the Hsc70-interacting protein (HIP).
We analyzed the cleavage pattern of HIP using two known activators of BID: granzyme B and caspase-8. In in vitro cleavage assays using recombinant proteins, human and rat HIP were cleaved by granzyme B. Furthermore, the granzyme B-mediated
cleavage site was mapped to the BID loop-like region of HIP by site-directed mutagenesis. This region was also the target
for caspase-8-mediated cleavage in rat HIP. However, human HIP was not proteolyzed by caspase-8, which probably reflects sequence
differences between human and rat HIP proteins at the P1′ position of the caspase-8 recognition sequence. To determine whether HIP is cleaved during apoptosis, human Jurkat T cells
were exposed to granzyme B and perforin. The results of these studies suggest that granzyme B-mediated loss of HIP expression
occurs in vivo, and in a coordinate fashion with loss of BID, pro-caspase-8 and pro-caspase-3. These data implicate the Hsp70 co-chaperone
HIP in the proteolytic cascade of some apoptotic pathways. 相似文献
10.
Huntingtin interacting protein 1 (HIP1) is a member of a family of proteins whose interaction with Huntingtin is critical to prevent cells from initiating apoptosis. HIP1, and related protein HIP12/1R, can also bind to clathrin and membrane phospholipids, and HIP12/1R links the CCV to the actin cytoskeleton. HIP1 and HIP12/1R interact with the clathrin light chain EED regulatory site and stimulate clathrin lattice assembly. Here, we report the X-ray structure of the coiled-coil domain of HIP1 (residues 482-586) that includes residues crucial for binding clathrin light chain. The dimeric HIP1 crystal structure is partially splayed open. The comparison of the HIP1 model with coiled-coil predictions revealed the heptad repeat in the dimeric trunk (S2 path) is offset relative to the register of the heptad repeat from the N-terminal portion (S1 path) of the molecule. Furthermore, surface analysis showed there is a third hydrophobic path (S3) running parallel with S1 and S2. We present structural evidence supporting a role for the S3 path as an interaction surface for clathrin light chain. Finally, comparative analysis suggests the mode of binding between sla2p and clathrin light chain may be different in yeast. 相似文献
11.
Sla2p Is Associated with the Yeast Cortical Actin Cytoskeleton via Redundant Localization Signals
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Shirley Yang M. Jamie T. V. Cope David G. Drubin 《Molecular biology of the cell》1999,10(7):2265-2283
Sla2p, also known as End4p and Mop2p, is the founding member of a widely conserved family of actin-binding proteins, a distinguishing feature of which is a C-terminal region homologous to the C terminus of talin. These proteins may function in actin cytoskeleton-mediated plasma membrane remodeling. A human homologue of Sla2p binds to huntingtin, the protein whose mutation results in Huntington's disease. Here we establish by immunolocalization that Sla2p is a component of the yeast cortical actin cytoskeleton. Deletion analysis showed that Sla2p contains two separable regions, which can mediate association with the cortical actin cytoskeleton, and which can provide Sla2p function. One localization signal is actin based, whereas the other signal is independent of filamentous actin. Biochemical analysis showed that Sla2p exists as a dimer in vivo. Two-hybrid analysis revealed two intramolecular interactions mediated by coiled-coil domains. One of these interactions appears to underlie dimer formation. The other appears to contribute to the regulation of Sla2p distribution between the cytoplasm and plasma membrane. The data presented are used to develop a model for Sla2p regulation and interactions. 相似文献
12.
《生物化学与生物物理学报:疾病的分子基础》2022,1868(2):166316
Mutations in superoxide dismutase 1 (SOD1) leading to the formation of intracellular protein aggregates cause amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder characterized by a selective degeneration of motor neurons. The ALS-linked mutant SOD1 emerged as a possible target for ubiquitin–proteasome system (UPS)-mediated degradation. We aimed to elucidate the role of huntingtin interaction protein 2 (HIP2), an E2 ubiquitin-conjugating enzyme, in the proteotoxicity of mutant SOD1 aggregates. We found that HIP2 interacts with mutant SOD1, but not wild-type SOD1, and is upregulated in response to mutant SOD1 expression. Upregulation of HIP2 protein was observed in the spinal cord of 16-week-old SOD1-G93A transgenic mice. HIP2 further modified mutant SOD1 proteins via K48-linked polyubiquitination and degraded mutant SOD1 proteins through the UPS. Upregulation of HIP2 protected cells from mutant SOD1-induced toxicity. Taken together, our findings demonstrate that HIP2 is a crucial regulator of quality control against the proteotoxicity of mutant SOD1. Our results suggest that modulating HIP2 may represent a novel therapeutic strategy for the treatment of ALS. 相似文献
13.
Huang K Yanai A Kang R Arstikaitis P Singaraja RR Metzler M Mullard A Haigh B Gauthier-Campbell C Gutekunst CA Hayden MR El-Husseini A 《Neuron》2004,44(6):977-986
In neurons, posttranslational modification by palmitate regulates the trafficking and function of signaling molecules, neurotransmitter receptors, and associated synaptic scaffolding proteins. However, the enzymatic machinery involved in protein palmitoylation has remained elusive. Here, using biochemical assays, we show that huntingtin (htt) interacting protein, HIP14, is a neuronal palmitoyl transferase (PAT). HIP14 shows remarkable substrate specificity for neuronal proteins, including SNAP-25, PSD-95, GAD65, synaptotagmin I, and htt. Conversely, HIP14 is catalytically invariant toward paralemmin and synaptotagmin VII. Exogenous HIP14 enhances palmitoylation-dependent vesicular trafficking of several acylated proteins in both heterologous cells and neurons. Moreover, interference with endogenous expression of HIP14 reduces clustering of PSD-95 and GAD65 in neurons. These findings define HIP14 as a mammalian palmitoyl transferase involved in the palmitoylation and trafficking of multiple neuronal proteins. 相似文献
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Daniel Feliciano Jarred J. Bultema Andrea L. Ambrosio Santiago M. Di Pietro 《Journal of visualized experiments : JoVE》2011,(47)
A major endocytic pathway initiates with the formation of clathrin-coated vesicles (CCVs) that transport cargo from the cell surface to endosomes1-6. CCVs are distinguished by a polyhedral lattice of clathrin that coats the vesicle membrane and serves as a mechanical scaffold. Clathrin coats are assembled during vesicle formation from individual clathrin triskelia , the soluble form of clathrin composed of three heavy and three light chain subunits7,8. Because the triskelion does not have the ability to bind to the membrane directly, clathrin-binding adaptors are critical to link the forming clathrin lattice to the membrane through association with lipids and/or membrane proteins9. Adaptors also package transmembrane protein cargo, such as receptors, and can interact with each other and with other components of the CCV formation machinery9.Over twenty clathrin adaptors have been described, several are involved in clathrin mediated endocytosis and others localize to the trans Golgi network or endosomes9. With the exception of HIP1R (yeast Sla2p), all known clathrin adaptors bind to the N-terminal -propeller domain of the clathrin heavy chain9. Clathrin adaptors are modular proteins consisting of folded domains connected by unstructured flexible linkers. Within these linker regions, short binding motifs mediate interactions with the clathrin N-terminal domain or other components of the vesicle formation machinery9. Two distinct clathrin-binding motifs have been defined: the clathrin-box and the W-box9. The consensus clathrin-box sequence was originally defined as L[L/I][D/E/N][L/F][D/E]10 but variants have been subsequently discovered11. The W-box conforms to the sequence PWxxW (where x is any residue).Sla1p (Synthetic Lethal with Actin binding protein-1) was originally identified as an actin associated protein and is necessary for normal actin cytoskeleton structure and dynamics at endocytic sites in yeast cells12. Sla1p also binds the NPFxD endocytic sorting signal and is critical for endocytosis of cargo bearing the NPFxD signal13,14. More recently, Sla1p was demonstrated to bind clathrin through a motif similar to the clathrin box, LLDLQ, termed a variant clathrin-box (vCB), and to function as an endocytic clathrin adaptor15. In addition, Sla1p has become a widely used marker for the endocytic coat in live cell fluorescence microscopy studies16. Here we use Sla1p as a model to describe approaches for adaptor-clathrin interaction studies. We focus on live cell fluorescence microscopy, GST-pull down, and co-immunoprecipitation methods.Download video file.(108M, mov) 相似文献
16.
Striking similarities between cytoskeletal assembly and the "nucleated polymerization" model of prion propagation suggest that similar or overlapping sets of proteins may assist in both processes. We show that the C-terminal domain of the yeast cytoskeletal assembly protein Sla1 (Sla1C) specifically interacts with the N-terminal prion-forming domain (Sup35N) of the yeast release factor Sup35 (eRF3) in the two-hybrid system. Sla1C and several other Sup35N-interacting proteins also exhibit two-hybrid interactions with the poly-Gln-expanded N-proximal fragment of human huntingtin, which promotes Huntington disease-associated aggregation. The Sup35N-Sla1C interaction is inhibited by Sup35N alterations that make Sup35 unable to propagate the [PSI(+)] state and by the absence of the chaperone protein Hsp104, which is essential for [PSI] propagation. In a Sla1(-) background, [PSI] curing by dimethylsulfoxide or excess Hsp104 is increased, while translational readthrough and de novo [PSI] formation induced by excess Sup35 or Sup35N are decreased. These data show that, in agreement with the proposed function of Sla1 during cytoskeletal formation, Sla1 assists in [PSI] formation and propagation, but is not required for these processes. Sla1(-) strains are sensitive to some translational inhibitors, and some sup35 mutants, obtained in a Sla1(-) background, are sensitive to Sla1, suggesting that the interaction between Sla1 and Sup35 proteins may play a role in the normal function of the translational apparatus. We hypothesize that Sup35N is involved in regulatory interactions with intracellular structural networks, and [PSI] prion may be formed as a by-product of this process. 相似文献
17.
Saint-Dic D Chang SC Taylor GS Provot MM Ross TS 《The Journal of biological chemistry》2001,276(24):21192-21198
It has been shown previously that the Huntingtin interacting protein 1 gene (HIP1) was fused to the platelet-derived growth factor beta receptor gene (PDGFbetaR) in leukemic cells of a patient with chronic myelomonocytic leukemia. This resulted in the expression of the chimeric HIP1/PDGFbetaR protein, which oligomerizes, is constitutively tyrosine-phosphorylated, and transforms the Ba/F3 murine hematopoietic cell line to interleukin-3-independent growth. Tyrosine phosphorylation of a 130-kDa protein (p130) correlates with transformation by HIP1/PDGFbetaR and related transforming mutants. We report here that the p130 band is immunologically related to the 125-kDa isoform of the Src homology 2-containing inositol 5-phosphatase, SHIP1. We have found that SHIP1 associates and colocalizes with the HIP1/PDGFbetaR fusion protein and related transforming mutants. These mutants include a mutant that has eight Src homology 2-binding phosphotyrosines mutated to phenylalanine. In contrast, SHIP1 does not associate with H/P(KI), the kinase-dead form of HIP1/PDGFbetaR. We also report that phosphorylation of SHIP1 by HIP1/PDGFbetaR does not change its 5-phosphatase-specific activity. This suggests that phosphorylation and possible PDGFbetaR-mediated sequestration of SHIP1 from its substrates (PtdIns(3,4,5)P(3) and Ins(1,3,4,5)P(4)) might alter the levels of these inositol-containing signal transduction molecules, resulting in activation of downstream effectors of cellular proliferation and/or survival. 相似文献
18.
Potyviral helper-component proteinase (HCpro) is a multifunctional protein exerting its cellular functions in interaction with putative host proteins. In this study, cellular protein partners of the HCpro encoded by Potato virus A (PVA) (genus Potyvirus) were screened in a potato leaf cDNA library using a yeast two-hybrid system. Two cellular proteins were obtained that interact specifically with PVA HCpro in yeast and in the two in vitro binding assays used. Both proteins are encoded by single-copy genes in the potato genome. Analysis of the deduced amino acid sequences revealed that one (HIP1) of the two HCpro interactors is a novel RING finger protein. The sequence of the other protein (HIP2) showed no resemblance to the protein sequences available from databanks and has known biological functions. 相似文献
19.
Sla1p is a functionally modular component of the yeast cortical actin cytoskeleton required for correct localization of both Rho1p-GTPase and Sla2p, a protein with talin homology
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Ayscough KR Eby JJ Lila T Dewar H Kozminski KG Drubin DG 《Molecular biology of the cell》1999,10(4):1061-1075
SLA1 was identified previously in budding yeast in a genetic screen for mutations that caused a requirement for the actin-binding protein Abp1p and was shown to be required for normal cortical actin patch structure and organization. Here, we show that Sla1p, like Abp1p, localizes to cortical actin patches. Furthermore, Sla1p is required for the correct localization of Sla2p, an actin-binding protein with homology to talin implicated in endocytosis, and the Rho1p-GTPase, which is associated with the cell wall biosynthesis enzyme beta-1,3-glucan synthase. Mislocalization of Rho1p in sla1 null cells is consistent with our observation that these cells possess aberrantly thick cell walls. Expression of mutant forms of Sla1p in which specific domains were deleted showed that the phenotypes associated with the full deletion are functionally separable. In particular, a region of Sla1p encompassing the third SH3 domain is important for growth at high temperatures, for the organization of cortical actin patches, and for nucleated actin assembly in a permeabilized yeast cell assay. The apparent redundancy between Sla1p and Abp1p resides in the C-terminal repeat region of Sla1p. A homologue of SLA1 was identified in Schizosaccharomyces pombe. Despite relatively low overall sequence homology, this gene was able to rescue the temperature sensitivity associated with a deletion of SLA1 in Saccharomyces cerevisiae. 相似文献
20.
Young FB Franciosi S Spreeuw A Deng Y Sanders S Tam NC Huang K Singaraja RR Zhang W Bissada N Kay C Hayden MR 《PloS one》2012,7(5):e36315
Huntingtin Interacting Protein 14 (HIP14) is a palmitoyl acyl transferase (PAT) that was first identified due to altered interaction with mutant huntingtin, the protein responsible for Huntington Disease (HD). HIP14 palmitoylates a specific set of neuronal substrates critical at the synapse, and downregulation of HIP14 by siRNA in vitro results in increased cell death in neurons. We previously reported that mice lacking murine Hip14 (Hip14-/-) share features of HD. In the current study, we have generated human HIP14 BAC transgenic mice and crossed them to the Hip14-/- model in order to confirm that the defects seen in Hip14-/- mice are in fact due to loss of Hip14. In addition, we sought to determine whether human HIP14 can provide functional compensation for loss of murine Hip14. We demonstrate that despite a relative low level of expression, as assessed via Western blot, BAC-derived human HIP14 compensates for deficits in neuropathology, behavior, and PAT enzyme function seen in the Hip14-/- model. Our findings yield important insights into HIP14 function in vivo. 相似文献