Microtubule Interacting And Transport Research Articles

Coding Mutations in Vacuolar Protein-Sorting 4 AAA+ ATPase Endosomal Sorting Complexes Required for Transport Protein Homologs Underlie bc-2 and New bc-4 Gene Conferring Resistance to Bean Common Mosaic Virus in Common Bean.

Bean common mosaic virus (BCMV) is a major disease in common bean (Phaseolus vulgaris L.). Host plant resistance is the most effective strategy to minimize crop damage against BCMV and the related Bean common mosaic necrosis virus (BCMNV). To facilitate breeding for resistance, we sought to identify candidate genes and develop markers for the bc-2 gene and the unknown gene with which it interacts. Genome-wide association study (GWAS) of the Durango Diversity Panel (DDP) identified a peak region for bc-2 on chromosome Pv11. Haplotype mapping narrowed the bc-2 genomic interval and identified Phvul.011G092700, a vacuolar protein-sorting 4 (Vps4) AAA+ ATPase endosomal sorting complexes required for transport (ESCRT) protein, as the bc-2 candidate gene. The race Durango Phvul.011G092700 gene model, bc-2[UI 111], contains a 10-kb deletion, while the race Mesoamerican bc-2[Robust] consists of a single nucleotide polymorphism (SNP) deletion. Each mutation introduces a premature stop codon, and they exhibit the same interaction with the pathogroups (PGs) tested. Phvul.005G125100, another Vps4 AAA+ ATPase ESCRT protein, was identified as the candidate gene for the new recessive bc-4 gene, and the recessive allele is likely an amino acid substitution in the microtubule interacting and transport (MIT) domain. The two Vps4 AAA+ ATPase ESCRT proteins exhibit high similarity to the Zym Cucsa.385040 candidate gene associated with recessive resistance to Zucchini yellow mosaic virus in cucumber. bc-2 alone has no resistance effect but, when combined with bc-4, provides resistance to BCMV (except PG-V) but not BCMNV, and, when combined with bc-ud, provides resistance to BCMV (except BCMV PG-VII) and BCMNV. So instead of different resistance alleles (i.e., bc-2 and bc-22), there is only bc-2 with a differential reaction based on whether it is combined with bc-4 or bc-ud, which are tightly linked in repulsion. The new tools and enhanced understanding of this host-virus pathogen interaction will facilitate breeding common beans for resistance to BCMV and BCMNV.

NRBF2 Regulates the Chemoresistance of Small Cell Lung Cancer by Interacting With the P62 Protein in the Autophagy Process

SUMMARY Reversing chemotherapy resistance in small cell lung cancer (SCLC) is crucial to improve patient prognosis. It is important to elucidate the mechanisms of chemoresistance and determine therapeutic targets for SCLC. The present study aims to investigate the underlying mechanisms in SCLC chemoresistance. We screen that nuclear receptor binding factor 2 (NRBF2) is a poor prognostic factor in SCLC. A correlation was identified between high NRBF2 expression and poor overall survival in the clinical local-SCLC cohort. The effects of NRBF2 on chemoresistance were determined in SCLC using a patient-derived xenograft model. The effect and underlying molecular mechanisms of NRBF2 in the autophagy process in SCLC were examined. NRBF2 positively regulated autophagy, leading to drug resistance in SCLC. The microtubule interacting and transport (MIT) domain of NRBF2 directly interacted with the PB1 domain of P62, a core autophagy marker. This interaction increased autophagic P62 body formation, revealing the regulatory role of NRBF2 in autophagy. Notably, NRBF2 was directly modulated by the transcription factor XRCC6, likely explaining the differential NRBF2 expression in chemosensitive and chemoresistant SCLC. The MIT domain of NRBF2 interacts with the PB1 domain of P62 to regulate the autophagy process, resulting in SCLC chemoresistance. NRBF2 is likely a useful chemotherapy response marker and therapeutic target in SCLC.

The Rice SPOTTED LEAF4 (SPL4) Encodes a Plant Spastin That Inhibits ROS Accumulation in Leaf Development and Functions in Leaf Senescence.

Lesion mimic mutants (LMMs) are usually controlled by single recessive mutations that cause the formation of necrotic lesions without pathogen invasion. These genetic defects are useful to reveal the regulatory mechanisms of defense-related programmed cell death in plants. Molecular evidence has been suggested that some of LMMs are closely associated with the regulation of leaf senescence in rice (Oryza sativa). Here, we characterized the mutation underlying spotted leaf4 (spl4), which results in lesion formation and also affects leaf senescence in rice. Map-based cloning revealed that the γ ray-induced spl4-1 mutant has a single base substitution in the splicing site of the SPL4 locus, resulting in a 13-bp deletion within the encoded microtubule-interacting-and-transport (MIT) spastin protein containing an AAA-type ATPase domain. The T-DNA insertion spl4-2 mutant exhibited spontaneous lesions similar to those of the spl4-1 mutant, confirming that SPL4 is responsible for the LMM phenotype. In addition, both spl4 mutants exhibited delayed leaf yellowing during dark-induced or natural senescence. Western blot analysis of spl4 mutant leaves suggested possible roles for SPL4 in the degradation of photosynthetic proteins. Punctate signals of SPL4-fused fluorescent proteins were detected in the cytoplasm, similar to the cellular localization of animal spastin. Based on these findings, we propose that SPL4 is a plant spastin that is involved in multiple aspects of leaf development, including senescence.

Crystallization and Biophysical Approaches for Studying the Interactions Between the Vps4-MIT Domain and ESCRT-III Proteins.

The AAA ATPase Vps4 disassembles the ESCRT complex from the endosomal membrane. Vps4 contains an N-terminal MIT (microtubule interacting and transport) domain and a C-terminal catalytic domain. The MIT domain binds to MIMs (MIT-interacting motifs), which exist at the C-terminus of ESCRT-III proteins, with a dissociation constant in the micromolar range. Five MIMs have been identified by structural and biophysical methods to date, and the recognition motifs have been refined. Among biophysical approaches used to analyze protein interactions, surface plasmon resonance (SPR) analysis is often suitable for weak interactions, and fluorescence-binding assay has an advantage in terms of sensitivity. We have introduced protein modification tags into the N-terminus of proteins with bacterial expression vectors for biotinylation and FlAsH (fluorescein arsenical hairpin binder) fluorescent labeling. Here, we describe how to purify the MIT domain of Vps4 and the MIMs of ESCRT-III proteins and how to conduct crystallography, SPR, and fluorescence-binding assays.

A Novel Mechanism of Regulating the ATPase VPS4 by Its Cofactor LIP5 and the Endosomal Sorting Complex Required for Transport (ESCRT)-III Protein CHMP5

Disassembly of the endosomal sorting complex required for transport (ESCRT) machinery from biological membranes is a critical final step in cellular processes that require the ESCRT function. This reaction is catalyzed by VPS4, an AAA-ATPase whose activity is tightly regulated by a host of proteins, including LIP5 and the ESCRT-III proteins. Here, we present structural and functional analyses of molecular interactions between human VPS4, LIP5, and the ESCRT-III proteins. The N-terminal domain of LIP5 (LIP5NTD) is required for LIP5-mediated stimulation of VPS4, and the ESCRT-III protein CHMP5 strongly inhibits the stimulation. Both of these observations are distinct from what was previously described for homologous yeast proteins. The crystal structure of LIP5NTD in complex with the MIT (microtubule-interacting and transport)-interacting motifs of CHMP5 and a second ESCRT-III protein, CHMP1B, was determined at 1 Å resolution. It reveals an ESCRT-III binding induced moderate conformational change in LIP5NTD, which results from insertion of a conserved CHMP5 tyrosine residue (Tyr(182)) at the core of LIP5NTD structure. Mutation of Tyr(182) partially relieves the inhibition displayed by CHMP5. Together, these results suggest a novel mechanism of VPS4 regulation in metazoans, where CHMP5 functions as a negative allosteric switch to control LIP5-mediated stimulation of VPS4.

Structural Basis of Molecular Recognition between ESCRT-III-like Protein Vps60 and AAA-ATPase Regulator Vta1 in the Multivesicular Body Pathway

The AAA-ATPase Vps4 is critical for function of the multivesicular body sorting pathway, which impacts cellular phenomena ranging from receptor down-regulation to viral budding to cytokinesis. Vps4 activity is stimulated by the interaction between Vta1 and Vps60, but the structural basis for this interaction is unclear. The fragment Vps60(128-186) was reported to display the full activity of Vps60. Vta1 interacts with Vps60 using its N-terminal domain (Vta1NTD). In this work, the structure of Vps60(128-186) in complex with Vta1NTD was determined using NMR techniques, demonstrating a novel recognition mode of the microtubule-interacting and transport (MIT) domain in which Vps60(128-186) interacts with Vta1NTD through helices α4' and α5', extending over Vta1NTD MIT2 domain helices 1-3. The Vps60 binding does not result in Vta1 conformational changes, further revealing the fact that Vps4 ATPase is enhanced by the interaction between Vta1 and Vps60 in an unanticipated manner.

Calpain-7 binds to CHMP1B at its second α-helical region and forms a ternary complex with IST1

Some intracellular proteins involved in the endosomal sorting complex required for transport (ESCRT) system have microtubule interacting and transport (MIT) domains and bind to ESCRT-III protein family members named charged multivesicular body proteins (CHMPs) at their C-terminal regions containing MIT-interacting motifs (MIMs). While two types of MIMs (MIM1 and MIM2) have been reported, CHMP1B has MIM1 and IST1 has both MIM1 and MIM2. Previously, we demonstrated that CHMP1B and IST1 directly interacted with a tandem repeat of MIT domains of calpain-7 (CL7MIT) and that autolytic activity of calpain-7 was enhanced by IST1 in vitro but not by overexpression of IST1 in HEK293T cells. In this study, we detected enhancement of autolysis of mGFP-fused calpain-7 by coexpression with CHMP1B and observed further activation by additional coexpression of IST1 in HEK293T cells. We found that CL7MIT interacted with the second α-helical region of CHMP1B but not with the canonical C-terminal region containing MIM1 in vitro. Co-immunoprecipitation assays demonstrated that the interaction between CL7MIT and CHMP1B and between CL7MIT and IST1 became stronger when IST1 or CHMP1B was additionally coexpressed, suggesting formation of ternary complex of calpain-7, IST1 and CHMP1B. Moreover, subcellular fractionation analyses revealed increase of calpain-7 in membrane/organelle fractions by concomitant overexpression of these ESCRT-III family member proteins.

ERBB2 is a target for USP8-mediated deubiquitination

Overexpression and poor downregulation of ErbB receptor tyrosine kinases are associated with enhanced signaling and tumorigenesis. Attenuation of EGF-receptor (EGFR) signaling is mediated by endocytosis and ubiquitination by the E3-ligase Cbl. En route to lysosomes, but before incorporation of the EGFR into internal vesicles of MVBs, the EGFR undergoes Usp8-mediated deubiquitination. ErbB2 displays enhanced recycling back to the cell surface, and therefore we hypothesized that Usp8 is not part of the ErbB2 trafficking pathway. Here, we demonstrate, in the context of a chimeric EGFR-ErbB2 receptor, that (i) EGF induces pY1091 Cbl binding site-dependent K63-polyubiquitination of EGFR-ErbB2, (ii) Cbl is tyrosine phosphorylated upon stimulation of EGFR-ErbB2 wt and Y1091F mutant receptor, (iii) EGF-induced activation of EGFR-ErbB2 induces Usp8 tyrosine phosphorylation, and (iv) ubiquitination of the EGFR-ErbB2 wt and Y1091F mutant is enhanced upon coexpression of catalytically inactive Usp8-C748A in the presence and absence of EGF. We further show that Usp8 tyrosine phosphorylation upon stimulation of EGFR-ErbB2 is (a) independent of Y1091, (b) dependent on Src- and EGFR-ErbB2-kinase activity, (c) enhanced upon coexpression of Usp8-C748A, and (d) partly dependent on the Microtubule Interacting and Transport (MIT) domain of Usp8. Our findings demonstrate that Usp8 is part of the ErbB2 endosomal trafficking pathway.

Autolytic activity of human calpain 7 is enhanced by ESCRT‐III‐related protein IST1 through MIT–MIM interaction

Calpain 7, a mammalian ortholog of yeast Cpl1/Rim13 and fungal PalB, is an atypical calpain that lacks a penta-EF-hand domain. Previously, we reported that a region containing a tandem repeat of microtubule-interacting and transport (MIT) domains in calpain 7 interacts with a subset of endosomal sorting complex required for transport (ESCRT)-III-related proteins, suggesting involvement of calpain 7 in the ESCRT system. Although yeast and fungal calpains are thought to be involved in alkaline adaptation via limited proteolysis of specific transcription factors, proteolytic activity of calpain 7 has not been demonstrated yet. In this study, we investigated the interaction between calpain 7 and a newly reported ESCRT-III family member, increased sodium tolerance-1 (IST1), which possesses two different types of MIT-interacting motifs (MIM1 and MIM2). We found that glutathione-S-transferase (GST)-fused tandem MIT domains of calpain 7 (calpain 7MIT) pulled down FLAG-tagged IST1 expressed in HEK293T cells. Coimmunoprecipitation assays with various deletion or point mutants of epitope-tagged calpain 7 and IST1 revealed that both repetitive MIT domains and MIMs are required for efficient interaction. Direct MIT-MIM binding was confirmed by a pulldown experiment with GST-fused IST1 MIM and purified recombinant calpain 7MIT. Furthermore, we found that the GST-MIM protein enhances the autolysis of purified Strep-tagged monomeric green fluorescent protein (mGFP)-fused calpain 7 (mGFP-calpain 7-Strep). The autolysis was almost completely abolished by 10 mmN-ethylmaleimide but only partially inhibited by 1 mm leupeptin or E-64. The putative catalytic Cys290-substituted mutant (mGFP-calpain 7(C290S)-Strep) showed no autolytic activity. These results demonstrate for the first time that human calpain 7 is proteolytically active, and imply that calpain 7 is activated in the ESCRT system.

Ab initio protein modelling reveals novel human MIT domains

Database searches can fail to detect all truly homologous sequences, particularly when dealing with short, highly sequence diverse protein families. Here, using microtubule interacting and transport (MIT) domains as an example, we have applied an approach of profile–profile matching followed by ab initio structure modelling to the detection of true homologues in the borderline significant zone of database searches. Novel MIT domains were confidently identified in USP54, containing an apparently inactive ubiquitin carboxyl-terminal hydrolase domain, a katanin-like ATPase KATNAL1, and an uncharacterized protein containing a VPS9 domain. As a proof of principle, we have confirmed the novel MIT annotation for USP54 by in vitro profiling of binding to CHMP proteins. Structured summary USP8 binds: CHMPs 1A 1B 2A 2B 4C USP54 binds: CHMPs 1B 2A 2B 4C 6

Evolution and assembly of ESCRTs

The AAA (ATPase associated with various cellular activities) proteins participate in membrane trafficking, organelle biogenesis, DNA replication, intracellular locomotion, cytoskeletal remodelling, protein folding and proteolysis. The AAA Vps (vacuolar protein sorting) 4 is central to traffic to lysosomes, retroviral budding and mammalian cell division. It dissociates ESCRTs (endosomal sorting complexes required for transport) from endosomal membranes, enabling their recycling to the cytosol, and plays a role in fission of intraluminal vesicles within MVBs (multivesicular bodies). The mechanism of Vps4-catalysed disassembly of ESCRT networks is unknown; however, it requires interaction between Vps4 and ESCRT-III subunits. The 30 C-terminal residues of Vps2 and Vps46 (Did2) subunits are both necessary and sufficient for interaction with the Vps4 N-terminal MIT (microtubule-interacting and transport) domain, and the crystal structure of the Vps2 C-terminus in a complex with the Vps4 MIT domain shows that MIT helices alpha2 and alpha3 recognize a (D/E)XXLXXRLXXL(K/R) MIM (MIT-interacting motif). These Vps2-MIT interactions are essential for vacuolar sorting and for Vps4-catalysed disassembly of ESCRT-III networks in vitro. Electron microscopy of ESCRT-III filaments assembled in vitro has enabled us to identify surfaces of the Vps24 subunit that are critical for protein sorting in vivo. The ESCRT-III-Vps4 interaction predates the divergence of Archaea and Eukarya. The Crenarchaea have three classes of ESCRT-III-like subunits, and one of these subunits interacts with an archaeal Vps4-like protein in a manner closely related to the human Vps4-human ESCRT-III subunit Vps20 interaction. This archaeal Vps4-ESCRT-III interaction appears to have a fundamental role in cell division in the Crenarchaea.

Essential Role of hIST1 in Cytokinesis

The last steps of multivesicular body (MVB) formation, human immunodeficiency virus (HIV)-1 budding and cytokinesis require a functional endosomal sorting complex required for transport (ESCRT) machinery to facilitate topologically equivalent membrane fission events. Increased sodium tolerance (IST) 1, a new positive modulator of the ESCRT pathway, has been described recently, but an essential function of this highly conserved protein has not been identified. Here, we describe the previously uncharacterized KIAA0174 as the human homologue of IST1 (hIST1), and we report its conserved interaction with VPS4, CHMP1A/B, and LIP5. We also identify a microtubule interacting and transport (MIT) domain interacting motif (MIM) in hIST1 that is necessary for its interaction with VPS4, LIP5 and other MIT domain-containing proteins, namely, MITD1, AMSH, UBPY, and Spastin. Importantly, hIST1 is essential for cytokinesis in mammalian cells but not for HIV-1 budding, thus providing a novel mechanism of functional diversification of the ESCRT machinery. Last, we show that the hIST1 MIM activity is essential for cytokinesis, suggesting possible mechanisms to explain the role of hIST1 in the last step of mammalian cell division.

Structural Basis for Autoinhibition of ESCRT-III CHMP3

Endosomal sorting complexes required for transport (ESCRT-0, ESCRT-I, ESCRT-II, and ESCRT-III) are selectively recruited to cellular membranes to exert their function in diverse processes, such as multivesicular body biogenesis, enveloped virus budding, and cytokinesis. ESCRT-III is composed of members of the charged multivesicular body protein (CHMP) family—cytosolic proteins that are targeted to membranes via yet unknown signals. Membrane targeting is thought to result in a membrane-associated protein network that presumably acts at a late budding step. Here we provide structural evidence based on small-angle X-ray scattering data that ESCRT-III CHMP3 can adopt two conformations in solution: a closed globular form that most likely represents the cytosolic conformation and an open extended conformation that might represent the activated form of CHMP3. Both the closed and open conformations of CHMP3 interact with AMSH with high affinity. Although the C-terminal region of CHMP3 is required for AMSH interaction, a peptide thereof reveals only weak binding to AMSH, suggesting that other regions of CHMP3 contribute to the high-affinity interaction. Thus, AMSH, including its MIT (microtubule interacting and transport) domain, interacts with ESCRT-III CHMP3 differently from reported Vps4 MIT domain–CHMP protein interactions.

Cryo-EM Structure of Dodecameric Vps4p and Its 2:1 Complex with Vta1p

The type I AAA (ATPase associated with a variety of cellular activities) ATPase Vps4 and its co-factor Vta1p/LIP5 function in membrane remodeling events that accompany cytokinesis, multivesicular body biogenesis, and retrovirus budding, apparently by driving disassembly and recycling of membrane-associated ESCRT (endosomal sorting complex required for transport)-III complexes. Here, we present electron cryomicroscopy reconstructions of dodecameric yeast Vps4p complexes with and without their microtubule interacting and transport (MIT) N-terminal domains and Vta1p co-factors. The ATPase domains of Vps4p form a bowl-like structure composed of stacked hexameric rings. The two rings adopt dramatically different conformations, with the “upper” ring forming an open assembly that defines the sides of the bowl and the lower ring forming a closed assembly that forms the bottom of the bowl. The N-terminal MIT domains of the upper ring localize on the symmetry axis above the cavity of the bowl, and the binding of six extended Vta1p monomers causes additional density to appear both above and below the bowl. The structures suggest models in which Vps4p MIT and Vta1p domains engage ESCRT-III substrates above the bowl and help transfer them into the bowl to be pumped through the center of the dodecameric assembly.

Structural basis for selective recognition of ESCRT-III by the AAA ATPase Vps4

The AAA+ ATPases are essential for various activities such as membrane trafficking, organelle biogenesis, DNA replication, intracellular locomotion, cytoskeletal remodelling, protein folding and proteolysis. The AAA ATPase Vps4, which is central to endosomal traffic to lysosomes, retroviral budding and cytokinesis, dissociates ESCRT complexes (the endosomal sorting complexes required for transport) from membranes. Here we show that, of the six ESCRT--related subunits in yeast, only Vps2 and Did2 bind the MIT (microtubule interacting and transport) domain of Vps4, and that the carboxy-terminal 30 residues of the subunits are both necessary and sufficient for interaction. We determined the crystal structure of the Vps2 C terminus in a complex with the Vps4 MIT domain, explaining the basis for selective ESCRT-III recognition. MIT helices alpha2 and alpha3 recognize a (D/E)xxLxxRLxxL(K/R) motif, and mutations within this motif cause sorting defects in yeast. Our crystal structure of the amino-terminal domain of an archaeal AAA ATPase of unknown function shows that it is closely related to the MIT domain of Vps4. The archaeal ATPase interacts with an archaeal ESCRT-III-like protein even though these organisms have no endomembrane system, suggesting that the Vps4/ESCRT-III partnership is a relic of a function that pre-dates the divergence of eukaryotes and Archaea.

The MIT Domain of UBPY Constitutes a CHMP Binding and Endosomal Localization Signal Required for Efficient Epidermal Growth Factor Receptor Degradation

We have identified and characterized a Microtubule Interacting and Transport (MIT) domain at the N terminus of the deubiquitinating enzyme UBPY/USP8. In common with other MIT-containing proteins such as AMSH and VPS4, UBPY can interact with CHMP proteins, which are known to regulate endosomal sorting of ubiquitinated receptors. Comparison of binding preferences for the 11 members of the human CHMP family between the UBPY MIT domain and another ubiquitin isopeptidase, AMSH, reveals common interactions with CHMP1A and CHMP1B but a distinct selectivity of AMSH for CHMP3/VPS24, a core subunit of the ESCRT-III complex, and UBPY for CHMP7. We also show that in common with AMSH, UBPY deubiquitinating enzyme activity can be stimulated by STAM but is unresponsive to its cognate CHMPs. The UBPY MIT domain is dispensable for its catalytic activity but is essential for its localization to endosomes. This is functionally significant as an MIT-deleted UBPY mutant is unable to rescue its binding partner STAM from proteasomal degradation or reverse a block to epidermal growth factor receptor degradation imposed by small interfering RNA-mediated depletion of UBPY.

ESCRT-III recognition by VPS4 ATPases

The ESCRT (endosomal sorting complex required for transport) pathway is required for terminal membrane fission events in several important biological processes, including endosomal intraluminal vesicle formation, HIV budding and cytokinesis. VPS4 ATPases perform a key function in this pathway by recognizing membrane-associated ESCRT-III assemblies and catalysing their disassembly, possibly in conjunction with membrane fission. Here we show that the microtubule interacting and transport (MIT) domains of human VPS4A and VPS4B bind conserved sequence motifs located at the carboxy termini of the CHMP1-3 class of ESCRT-III proteins. Structures of VPS4A MIT-CHMP1A and VPS4B MIT-CHMP2B complexes reveal that the C-terminal CHMP motif forms an amphipathic helix that binds in a groove between the last two helices of the tetratricopeptide-like repeat (TPR) of the VPS4 MIT domain, but in the opposite orientation to that of a canonical TPR interaction. Distinct pockets in the MIT domain bind three conserved leucine residues of the CHMP motif, and mutations that inhibit these interactions block VPS4 recruitment, impair endosomal protein sorting and relieve dominant-negative VPS4 inhibition of HIV budding. Thus, our studies reveal how the VPS4 ATPases recognize their CHMP substrates to facilitate the membrane fission events required for the release of viruses, endosomal vesicles and daughter cells.

Structure and ESCRT-III protein interactions of the MIT domain of human VPS4A.

The VPS4 AAA ATPases function both in endosomal vesicle formation and in the budding of many enveloped RNA viruses, including HIV-1. VPS4 proteins act by binding and catalyzing release of the membrane-associated ESCRT-III protein lattice, thereby allowing multiple rounds of protein sorting and vesicle formation. Here, we report the solution structure of the N-terminal VPS4A microtubule interacting and transport (MIT) domain and demonstrate that the VPS4A MIT domain binds the C-terminal half of the ESCRT-III protein, CHMP1B (Kd = 20 +/- 13 microM). The MIT domain forms an asymmetric three-helix bundle that resembles the first three helices in a tetratricopeptide repeat (TPR) motif. Unusual interhelical interactions are mediated by a series of conserved aromatic residues that form coiled-coil interactions between the second two helices and also pack against the conserved alanines that interdigitate between the first two helices. Mutational analyses revealed that a conserved leucine residue (Leu-64) on the third helix that would normally bind the fourth helix in an extended TPR is used to bind CHMP1B, raising the possibility that ESCRT-III proteins may bind by completing the TPR motif.