scholarly journals Structural insights into WHAMM-mediated cytoskeletal coordination during membrane remodeling

2012 ◽  
Vol 199 (1) ◽  
pp. 111-124 ◽  
Author(s):  
Qing-Tao Shen ◽  
Peter P. Hsiue ◽  
Charles V. Sindelar ◽  
Matthew D. Welch ◽  
Kenneth G. Campellone ◽  
...  
Keyword(s):  
Intracellular Transport ◽  
Coiled Coil ◽  
Tubular Structures ◽  
Terminal Portion ◽  
Actin Nucleation ◽  
Cellular Processes ◽  
Coiled Coil Region ◽  
Membrane Tubulation ◽  
Underlying Mechanisms ◽  
Structural Insights

The microtubule (MT) and actin cytoskeletons drive many essential cellular processes, yet fairly little is known about how their functions are coordinated. One factor that mediates important cross talk between these two systems is WHAMM, a Golgi-associated protein that utilizes MT binding and actin nucleation activities to promote membrane tubulation during intracellular transport. Using cryoelectron microscopy and other biophysical and biochemical approaches, we unveil the underlying mechanisms for how these activities are coordinated. We find that WHAMM bound to the outer surface of MT protofilaments via a novel interaction between its central coiled-coil region and tubulin heterodimers. Upon the assembly of WHAMM onto MTs, its N-terminal membrane-binding domain was exposed at the MT periphery, where it can recruit vesicles and remodel them into tubular structures. In contrast, MT binding masked the C-terminal portion of WHAMM and prevented it from promoting actin nucleation. These results give rise to a model whereby distinct MT-bound and actin-nucleating populations of WHAMM collaborate during membrane tubulation.

scholarly journals Peroxisomes move by hitchhiking on early endosomes using the novel linker protein PxdA

2015 ◽  
Author(s):  
John Salogiannis ◽  
Martin J. Egan ◽  
Samara L. Reck-Peterson
Keyword(s):  
Molecular Motors ◽  
Intracellular Transport ◽  
Coiled Coil ◽  
Leading Edge ◽  
Time Lapse ◽  
Early Endosome ◽  
Organelle Transport ◽  
The Novel ◽  
Coiled Coil Region ◽  
Early Endosomes

Eukaryotic cells use microtubule-based intracellular transport for the delivery of many subcellular cargos, including organelles. The canonical view of organelle transport is that organelles directly recruit molecular motors via cargo-specific adaptors. In contrast to this view, we show here that peroxisomes move by hitchhiking on early endosomes, an organelle that directly recruits the transport machinery. Using the filamentous fungus Aspergillus nidulans we find that hitchhiking is mediated by a novel endosome-associated linker protein, PxdA. PxdA is required for normal distribution and long-range movement of peroxisomes, but not early endosomes or nuclei. Using simultaneous time-lapse imaging we find that early endosome-associated PxdA localizes to the leading edge of moving peroxisomes. We identify a coiled-coil region within PxdA that is necessary and sufficient for early endosome localization and peroxisome distribution and motility. These results present a new mechanism of microtubule-based organelle transport where peroxisomes hitchhike on early endosomes and identify PxdA as the novel linker protein required for this coupling.

scholarly journals SVIP Is a Novel VCP/p97-interacting Protein Whose Expression Causes Cell Vacuolation

2003 ◽  
Vol 14 (1) ◽  
pp. 262-273 ◽  
Author(s):  
Masami Nagahama ◽  
Mie Suzuki ◽  
Yuko Hamada ◽  
Kiyotaka Hatsuzawa ◽  
Katsuko Tani ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Coiled Coil ◽  
Adaptor Proteins ◽  
Interacting Protein ◽  
Cellular Processes ◽  
Coiled Coil Region ◽  
Acid Protein ◽  
Extensive Cell ◽  
Dependent Protein ◽  
Amino Acid Protein

VCP/p97 is involved in a variety of cellular processes, including membrane fusion and ubiquitin-dependent protein degradation. It has been suggested that adaptor proteins such as p47 and Ufd1p confer functional versatility to VCP/p97. To identify novel adaptors, we searched for proteins that interact specifically with VCP/p97 by using the yeast two-hybrid system, and discovered a novel VCP/p97-interacting protein named smallVCP/p97-interactingprotein (SVIP). Rat SVIP is a 76-amino acid protein that contains two putative coiled-coil regions, and potential myristoylation and palmitoylation sites at the N terminus. Binding experiments revealed that the N-terminal coiled-coil region of SVIP, and the N-terminal and subsequent ATP-binding regions (ND1 domain) of VCP/p97, interact with each other. SVIP and previously identified adaptors p47 and ufd1p interact with VCP/p97 in a mutually exclusive manner. Overexpression of full-length SVIP or a truncated mutant did not markedly affect the structure of the Golgi apparatus, but caused extensive cell vacuolation reminiscent of that seen upon the expression of VCP/p97 mutants or polyglutamine proteins in neuronal cells. The vacuoles seemed to be derived from endoplasmic reticulum membranes. These results together suggest that SVIP is a novelVCP/p97 adaptor whose function is related to the integrity of the endoplasmic reticulum.

Functional interactions between ubiquitin E2 enzymes and TRIM proteins

2011 ◽  
Vol 434 (2) ◽  
pp. 309-319 ◽  
Author(s):  
Luisa M. Napolitano ◽  
Ellis G. Jaffray ◽  
Ronald T. Hay ◽  
Germana Meroni
Keyword(s):  
Coiled Coil ◽  
Specific Interactions ◽  
E3 Ligases ◽  
Cellular Processes ◽  
Coiled Coil Region ◽  
Physiological Relevance ◽  
Tripartite Motif ◽  
E2 Enzymes ◽  
Specific Reactions ◽  
Trim Proteins

The TRIM (tripartite motif) family of proteins is characterized by the presence of the tripartite motif module, composed of a RING domain, one or two B-box domains and a coiled-coil region. TRIM proteins are involved in many cellular processes and represent the largest subfamily of RING-containing putative ubiquitin E3 ligases. Whereas their role as E3 ubiquitin ligases has been presumed, and in several cases established, little is known about their specific interactions with the ubiquitin-conjugating E2 enzymes or UBE2s. In the present paper, we report a thorough screening of interactions between the TRIM and UBE2 families. We found a general preference of the TRIM proteins for the D and E classes of UBE2 enzymes, but we also revealed very specific interactions between TRIM9 and UBE2G2, and TRIM32 and UBE2V1/2. Furthermore, we demonstrated that the TRIM E3 activity is only manifest with the UBE2 with which they interact. For most specific interactions, we could also observe subcellular co-localization of the TRIM involved and its cognate UBE2 enzyme, suggesting that the specific selection of TRIM–UBE2 pairs has physiological relevance. Our findings represent the basis for future studies on the specific reactions catalysed by the TRIM E3 ligases to determine the fate of their targets.

scholarly journals KIF13A motors are regulated by Rab22A to function as weak dimers inside the cell

2021 ◽  
Vol 7 (6) ◽  
pp. eabd2054
Author(s):  
Nishaben M. Patel ◽  
Meenakshi Sundaram Aravintha Siva ◽  
Ruchi Kumari ◽  
Dipeshwari J. Shewale ◽  
Ashim Rai ◽  
...  
Keyword(s):  
Single Molecule ◽  
Regulatory Mechanism ◽  
Coiled Coil ◽  
Recycling Endosomes ◽  
Single Molecule Level ◽  
Cellular Processes ◽  
Membrane Tubulation ◽  
Guanosine Triphosphatase ◽  
Neck Coil ◽  
Unique Mechanism

Endocytic recycling is a complex itinerary, critical for many cellular processes. Membrane tubulation is a hallmark of recycling endosomes (REs), mediated by KIF13A, a kinesin-3 family motor. Understanding the regulatory mechanism of KIF13A in RE tubulation and cargo recycling is of fundamental importance but is overlooked. Here, we report a unique mechanism of KIF13A dimerization modulated by Rab22A, a small guanosine triphosphatase, during RE tubulation. A conserved proline between neck coil–coiled-coil (NC-CC1) domains of KIF13A creates steric hindrance, rendering the motors as inactive monomers. Rab22A plays an unusual role by binding to NC-CC1 domains of KIF13A, relieving proline-mediated inhibition and facilitating motor dimerization. As a result, KIF13A motors produce balanced motility and force against multiple dyneins in a molecular tug-of-war to regulate RE tubulation and homeostasis. Together, our findings demonstrate that KIF13A motors are tuned at a single-molecule level to function as weak dimers on the cellular cargo.

scholarly journals Cryo-EM of human Arp2/3 complexes provides structural insights into actin nucleation modulation by ARPC5 isoforms

2020 ◽  
Author(s):  
Ottilie von Loeffelholz ◽  
Andrew Purkiss ◽  
Luyan Cao ◽  
Svend Kjaer ◽  
Naoko Kogata ◽  
...  
Keyword(s):  
Actin Filament ◽  
Important Determinant ◽  
Complex Activity ◽  
Actin Nucleation ◽  
Cellular Processes ◽  
N Terminus ◽  
Nucleation Activity ◽  
Filament Networks ◽  
Structural Insights ◽  
Shed Light

AbstractThe Arp2/3 complex regulates many cellular processes by stimulating formation of branched actin filament networks. Because three of its seven subunits exist as two different isoforms, mammals produce a family of Arp2/3 complexes with different properties that may be suited to different physiological contexts. To shed light on how isoform diversification affects Arp2/3 function, we determined a 4.2 Å resolution cryo-EM structure of the most active human Arp2/3 complex containing ARPC1B and ARPC5L, and compared it with the structure of the least active ARPC1A-ARPC5-containing complex. The architecture of each isoform-specified Arp2/3 is the same. Strikingly, however, the N-terminal half of ARPC5L is partially disordered compared to ARPC5, suggesting that this region of ARPC5/ARPC5L is an important determinant of complex activity. Confirming this idea, the nucleation activity of Arp2/3 complexes containing hybrid ARPC5/ARPC5L subunits is higher when the ARPC5L N-terminus is present, thereby explaining activity differences between the different Arp2/3 complexes.

scholarly journals Structure of the human C9orf72-SMCR8 complex reveals a multivalent protein interaction architecture

PLoS Biology ◽  
2021 ◽  
Vol 19 (7) ◽  
pp. e3001344
Author(s):  
Julia Nörpel ◽  
Simone Cavadini ◽  
Andreas D. Schenk ◽  
Alexandra Graff-Meyer ◽  
Daniel Hess ◽  
...  
Keyword(s):  
Coiled Coil ◽  
Interacting Protein ◽  
Trimeric Complex ◽  
Cryo Electron Microscopy ◽  
Coiled Coil Region ◽  
C9orf72 Gene ◽  
Cellular Pathways ◽  
Underlying Mechanisms ◽  
Lateral Sclerosis ◽  
Dipeptide Repeat

A major cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) spectrum disorder is the hexanucleotide G4C2 repeat expansion in the first intron of the C9orf72 gene. Many underlying mechanisms lead to manifestation of disease that include toxic gain of function by repeat G4C2 RNAs, dipeptide repeat proteins, and a reduction of the C9orf72 gene product. The C9orf72 protein interacts with SMCR8 and WDR41 to form a trimeric complex and regulates multiple cellular pathways including autophagy. Here, we report the structure of the C9orf72-SMCR8 complex at 3.8 Å resolution using single-particle cryo-electron microscopy (cryo-EM). The structure reveals 2 distinct dimerization interfaces between C9orf72 and SMCR8 that involves an extensive network of interactions. Homology between C9orf72-SMCR8 and Folliculin-Folliculin Interacting Protein 2 (FLCN-FNIP2), a GTPase activating protein (GAP) complex, enabled identification of a key residue within the active site of SMCR8. Further structural analysis suggested that a coiled-coil region within the uDenn domain of SMCR8 could act as an interaction platform for other coiled-coil proteins, and its deletion reduced the interaction of the C9orf72-SMCR8 complex with FIP200 upon starvation. In summary, this study contributes toward our understanding of the biological function of the C9orf72-SMCR8 complex.

scholarly journals Peroxisomes move by hitchhiking on early endosomes using the novel linker protein PxdA

2016 ◽  
Vol 212 (3) ◽  
pp. 289-296 ◽  
Author(s):  
John Salogiannis ◽  
Martin J. Egan ◽  
Samara L. Reck-Peterson
Keyword(s):  
Molecular Motors ◽  
Intracellular Transport ◽  
Coiled Coil ◽  
Leading Edge ◽  
Time Lapse ◽  
Early Endosome ◽  
Organelle Transport ◽  
The Novel ◽  
Coiled Coil Region ◽  
Early Endosomes

Eukaryotic cells use microtubule-based intracellular transport for the delivery of many subcellular cargos, including organelles. The canonical view of organelle transport is that organelles directly recruit molecular motors via cargo-specific adaptors. In contrast with this view, we show here that peroxisomes move by hitchhiking on early endosomes, an organelle that directly recruits the transport machinery. Using the filamentous fungus Aspergillus nidulans we found that hitchhiking is mediated by a novel endosome-associated linker protein, PxdA. PxdA is required for normal distribution and long-range movement of peroxisomes, but not early endosomes or nuclei. Using simultaneous time-lapse imaging, we find that early endosome-associated PxdA localizes to the leading edge of moving peroxisomes. We identify a coiled-coil region within PxdA that is necessary and sufficient for early endosome localization and peroxisome distribution and motility. These results present a new mechanism of microtubule-based organelle transport in which peroxisomes hitchhike on early endosomes and identify PxdA as the novel linker protein required for this coupling.

scholarly journals Characterisation of class VI TRIM RING domains: linking RING activity to C-terminal domain identity

2019 ◽  
Vol 2 (3) ◽  
pp. e201900295 ◽  
Author(s):  
Rebecca V Stevens ◽  
Diego Esposito ◽  
Katrin Rittinger
Keyword(s):  
Solution Structure ◽  
Coiled Coil ◽  
E3 Ligase ◽  
Cellular Processes ◽  
Terminal Domain ◽  
Coiled Coil Region ◽  
Biochemical Characterisation ◽  
Ring Domains ◽  
Self Association ◽  
Terminal Domains

TRIM E3 ubiquitin ligases regulate multiple cellular processes, and their dysfunction is linked to disease. They are characterised by a conserved N-terminal tripartite motif comprising a RING, B-box domains, and a coiled-coil region, with C-terminal domains often mediating substrate recruitment. TRIM proteins are grouped into 11 classes based on C-terminal domain identity. Class VI TRIMs, TRIM24, TRIM33, and TRIM28, have been described as transcriptional regulators, a function linked to their C-terminal plant homeodomain and bromodomain, and independent of their ubiquitination activity. It is unclear whether E3 ligase activity is regulated in family members where the C-terminal domains function independently. Here, we provide a detailed biochemical characterisation of the RING domains of class VI TRIMs and describe the solution structure of the TRIM28 RING. Our study reveals a lack of activity of the isolated RING domains, which may be linked to the absence of self-association. We propose that class VI TRIMs exist in an inactive state and require additional regulatory events to stimulate E3 ligase activity, ensuring that associated chromatin-remodelling factors are not injudiciously degraded.

scholarly journals Coiled-Coil N21 of Hpa1 in Xanthomonas oryzae pv. oryzae Promotes Plant Growth, Disease Resistance and Drought Tolerance in Non-Hosts via Eliciting HR and Regulation of Multiple Defense Response Genes

2020 ◽  
Vol 22 (1) ◽  
pp. 203
Author(s):  
Zhao-Lin Ji ◽  
Mei-Hui Yu ◽  
Ya-Yan Ding ◽  
Jian Li ◽  
Feng Zhu ◽  
...  
Keyword(s):  
Drought Tolerance ◽  
Coiled Coil ◽  
Pharmaceutical Products ◽  
Full Length ◽  
Xanthomonas Oryzae ◽  
Disease Symptoms ◽  
Pathogenic Factors ◽  
Defense Response Genes ◽  
Underlying Mechanisms ◽  
Further Development

Acting as a typical harpin protein, Hpa1 of Xanthomonas oryzae pv. oryzae is one of the pathogenic factors in hosts and can elicit hypersensitive responses (HR) in non-hosts. To further explain the underlying mechanisms of its induced resistance, we studied the function of the most stable and shortest three heptads in the N-terminal coiled-coil domain of Hpa1, named N21Hpa1. Proteins isolated from N21-transgenic tobacco elicited HR in Xanthi tobacco, which was consistent with the results using N21 and full-length Hpa1 proteins expressed in Escherichia coli. N21-expressing tobacco plants showed enhanced resistance to tobacco mosaic virus (TMV) and Pectobacterium carotovora subsp. carotovora (Pcc). Spraying of a synthesized N21 peptide solution delayed the disease symptoms caused by Botrytis cinerea and Monilinia fructicola and promoted the growth and drought tolerance of plants. Further analysis indicated that N21 upregulated the expression of multiple plant defense-related genes, such as genes mediated by salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) signaling, and genes related to reactive oxygen species (ROS) biosynthesis. Further, the bioavailability of N21 peptide was better than that of full-length Hpa1Xoo. Our studies support the broad application prospects of N21 peptide as a promising succedaneum to biopesticide Messenger or Illite or other biological pharmaceutical products, and provide a basis for further development of biopesticides using proteins with similar structures.

scholarly journals Reggie/flotillin proteins are organized into stable tetramers in membrane microdomains

2007 ◽  
Vol 403 (2) ◽  
pp. 313-322 ◽  
Author(s):  
Gonzalo P. Solis ◽  
Maja Hoegg ◽  
Christina Munderloh ◽  
Yvonne Schrock ◽  
Edward Malaga-Trillo ◽  
...  
Keyword(s):  
T Cell Activation ◽  
Cell Activation ◽  
Coiled Coil ◽  
Cellular Prion Protein ◽  
Membrane Microdomains ◽  
Protein Assemblies ◽  
Cellular Processes ◽  
C Terminus ◽  
Functional Relevance ◽  
Interfering Rna

Reggie-1 and -2 proteins (flotillin-2 and -1 respectively) form their own type of non-caveolar membrane microdomains, which are involved in important cellular processes such as T-cell activation, phagocytosis and signalling mediated by the cellular prion protein and insulin; this is consistent with the notion that reggie microdomains promote protein assemblies and signalling. While it is generally known that membrane microdomains contain large multiprotein assemblies, the exact organization of reggie microdomains remains elusive. Using chemical cross-linking approaches, we have demonstrated that reggie complexes are composed of homo- and hetero-tetramers of reggie-1 and -2. Moreover, native reggie oligomers are indeed quite stable, since non-cross-linked tetramers are resistant to 8 M urea treatment. We also show that oligomerization requires the C-terminal but not the N-terminal halves of reggie-1 and -2. Using deletion constructs, we analysed the functional relevance of the three predicted coiled-coil stretches present in the C-terminus of reggie-1. We confirmed experimentally that reggie-1 tetramerization is dependent on the presence of coiled-coil 2 and, partially, of coiled-coil 1. Furthermore, since depletion of reggie-1 by siRNA (small interfering RNA) silencing induces proteasomal degradation of reggie-2, we conclude that the protein stability of reggie-2 depends on the presence of reggie-1. Our data indicate that the basic structural units of reggie microdomains are reggie homo- and hetero-tetramers, which are dependent on the presence of reggie-1.

Sign in / Sign up

Export Citation Format

Share Document