Ubiquitin and endocytic protein sorting

2005 ◽  
Vol 41 ◽  
pp. 81-98 ◽  
Author(s):  
Sylvie Urbé
Keyword(s):  
Membrane Proteins ◽  
Tyrosine Kinases ◽  
Mammalian Cells ◽  
Degradation Process ◽  
Deubiquitinating Enzymes ◽  
Endosomal Sorting ◽  
Binding Domains ◽  
C Terminus ◽  
Lysosomal Sorting ◽  
Ubiquitin Ligase E3

Ubiquitin plays a fundamental role not only in proteasome-mediated protein degradation but also in the targeting of membrane proteins for degradation inside the lysosome. Ubiquitination provides a key signal for endosomal sorting of membrane proteins into the MVB (multi-vesicular body), which delivers its cargo to the proteolytic interior of the lysosome. Attachment of single ubiquitin molecules, rather than ubiquitin chains, to one or multiple lysines of the cytoplasmic domains of many growth factor receptors, ion channels and other membrane transporters is sufficient to target these proteins to a complex sorting apparatus on the endosome. This machinery selects ubiquitinated proteins for lysosomal sorting through consecutive interactions with a variety of ubiquitin-binding domains. The major ubiquitin ligase (E3) responsible for ubiquitination in this pathway in yeast is the HECT [homologous to E6-AP (E6-associated protein) C-terminus]-ligase, Rsp5, whereas in mammalian cells the RING (really interesting new gene)-ligase Cbl has been implicated in the down-regulation of several RTKs (receptor tyrosine kinases). Ubiquitinated receptors can be rescued from degradation by the activity of DUBs (deubiquitinating enzymes), which may provide a proofreading mechanism that enhances the fidelity of this sorting and degradation process. DUBs also allow for recycling of the ubiquitin moieties from proteins prior to their final commitment to the MVB and lysosome interior.

scholarly journals ESCRT ubiquitin-binding domains function cooperatively during MVB cargo sorting

2009 ◽  
Vol 185 (2) ◽  
pp. 213-224 ◽  
Author(s):  
S. Brookhart Shields ◽  
Andrea J. Oestreich ◽  
Stanley Winistorfer ◽  
Doris Nguyen ◽  
Johanna A. Payne ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Structural Information ◽  
Multivesicular Bodies ◽  
Endosomal Sorting ◽  
Binding Domains ◽  
Ubiquitin Binding ◽  
Cargo Sorting

Ubiquitin (Ub) sorting receptors facilitate the targeting of ubiquitinated membrane proteins into multivesicular bodies (MVBs). Ub-binding domains (UBDs) have been described in several endosomal sorting complexes required for transport (ESCRT). Using available structural information, we have investigated the role of the multiple UBDs within ESCRTs during MVB cargo selection. We found a novel UBD within ESCRT-I and show that it contributes to MVB sorting in concert with the known UBDs within the ESCRT complexes. These experiments reveal an unexpected level of coordination among the ESCRT UBDs, suggesting that they collectively recognize a diverse set of cargo rather than act sequentially at discrete steps.

scholarly journals ALIX binds a YPX3L motif of the GPCR PAR1 and mediates ubiquitin-independent ESCRT-III/MVB sorting

2012 ◽  
Vol 197 (3) ◽  
pp. 407-419 ◽  
Author(s):  
Michael R. Dores ◽  
Buxin Chen ◽  
Huilan Lin ◽  
Unice J.K. Soh ◽  
May M. Paing ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Interacting Protein ◽  
Kinase Substrate ◽  
Endosomal Sorting ◽  
Ubiquitin Binding ◽  
Lysosomal Sorting ◽  
Protease Activated Receptor 1 ◽  
Signaling Receptors ◽  
G Protein Coupled ◽  
Hepatocyte Growth

The sorting of signaling receptors to lysosomes is an essential regulatory process in mammalian cells. During degradation, receptors are modified with ubiquitin and sorted by endosomal sorting complex required for transport (ESCRT)–0, –I, –II, and –III complexes into intraluminal vesicles (ILVs) of multivesicular bodies (MVBs). However, it remains unclear whether a single universal mechanism mediates MVB sorting of all receptors. We previously showed that protease-activated receptor 1 (PAR1), a G protein–coupled receptor (GPCR) for thrombin, is internalized after activation and sorted to lysosomes independent of ubiquitination and the ubiquitin-binding ESCRT components hepatocyte growth factor–regulated tyrosine kinase substrate and Tsg101. In this paper, we report that PAR1 sorted to ILVs of MVBs through an ESCRT-III–dependent pathway independent of ubiquitination. We further demonstrate that ALIX, a charged MVB protein 4–ESCRT-III interacting protein, bound to a YPX3L motif of PAR1 via its central V domain to mediate lysosomal degradation. This study reveals a novel MVB/lysosomal sorting pathway for signaling receptors that bypasses the requirement for ubiquitination and ubiquitin-binding ESCRTs and may be applicable to a subset of GPCRs containing YPXnL motifs.

scholarly journals Type II transmembrane domain hydrophobicity dictates the cotranslational dependence for inversion

2014 ◽  
Vol 25 (21) ◽  
pp. 3363-3374 ◽  
Author(s):  
Dan Dou ◽  
Diogo V. da Silva ◽  
Johan Nordholm ◽  
Hao Wang ◽  
Robert Daniels
Keyword(s):  
Amino Acids ◽  
Membrane Proteins ◽  
Mammalian Cells ◽  
Influenza A ◽  
Transmembrane Domain ◽  
Membrane Insertion ◽  
Type Ii ◽  
C Terminus ◽  
Insertion Process ◽  
Positively Charged

Membrane insertion by the Sec61 translocon in the endoplasmic reticulum (ER) is highly dependent on hydrophobicity. This places stringent hydrophobicity requirements on transmembrane domains (TMDs) from single-spanning membrane proteins. On examining the single-spanning influenza A membrane proteins, we found that the strict hydrophobicity requirement applies to the Nout-CinHA and M2 TMDs but not the Nin-CoutTMDs from the type II membrane protein neuraminidase (NA). To investigate this discrepancy, we analyzed NA TMDs of varying hydrophobicity, followed by increasing polypeptide lengths, in mammalian cells and ER microsomes. Our results show that the marginally hydrophobic NA TMDs (ΔGapp> 0 kcal/mol) require the cotranslational insertion process for facilitating their inversion during translocation and a positively charged N-terminal flanking residue and that NA inversion enhances its plasma membrane localization. Overall the cotranslational inversion of marginally hydrophobic NA TMDs initiates once ∼70 amino acids past the TMD are synthesized, and the efficiency reaches 50% by ∼100 amino acids, consistent with the positioning of this TMD class in type II human membrane proteins. Inversion of the M2 TMD, achieved by elongating its C-terminus, underscores the contribution of cotranslational synthesis to TMD inversion.

scholarly journals Roles for ER:endosome membrane contact sites in ligand-stimulated intraluminal vesicle formation

2018 ◽  
Vol 46 (5) ◽  
pp. 1055-1062 ◽  
Author(s):  
Louise H. Wong ◽  
Emily R. Eden ◽  
Clare E. Futter
Keyword(s):  
Membrane Proteins ◽  
Mammalian Cells ◽  
Tyrosine Phosphatase ◽  
Endosomal Sorting ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Membrane Contacts ◽  
Epidermal Growth ◽  
Membrane Contact ◽  
Multivesicular Endosomes

Multivesicular endosomes/bodies (MVBs) sort membrane proteins between recycling and degradative pathways. Segregation of membrane proteins onto intraluminal vesicles (ILVs) of MVBs removes them from the recycling pathway and facilitates their degradation following fusion of MVBs with lysosomes. Sorting of many cargos onto ILVs depends on the ESCRT (Endosomal Sorting Complex Required for Transport) machinery, although ESCRT-independent mechanisms also exist. In mammalian cells, efficient sorting of ligand-stimulated epidermal growth factor receptors onto ILVs also depends on the tyrosine phosphatase, PTP1B, an ER-localised enzyme that interacts with endosomal targets at membrane contacts between MVBs and the ER. This review focuses on the potential roles played by ER:MVB membrane contact sites in regulating ESCRT-dependent ILV formation.

scholarly journals His Domain Protein Tyrosine Phosphatase and Rabaptin-5 couple endo-lysosomal sorting of EGFR with endosomal maturation

2021 ◽  
Author(s):  
Gabrielle Parkinson ◽  
Peristera Roboti ◽  
Ling Zhang ◽  
Sandra Taylor ◽  
Philip Woodman
Keyword(s):  
Protein Tyrosine Phosphatase ◽  
Tyrosine Phosphatase ◽  
Multivesicular Body ◽  
Nucleotide Exchange ◽  
Protein Tyrosine ◽  
Endosomal Sorting ◽  
Binding Domains ◽  
Nucleotide Exchange Factor ◽  
Lysosomal Sorting ◽  
Domain Protein

His Domain Protein Tyrosine Phosphatase (HD-PTP) collaborates with Endosomal Sorting Complexes Required for Transport (ESCRTs) to sort endosomal cargo into intralumenal vesicles, forming the multivesicular body. Completion of multivesicular body sorting is accompanied by maturation of the endosome into a late endosome, an event that requires inactivation of the early endosomal GTPase, Rab5. Here we show that HD-PTP links ESCRT function with endosomal maturation. HD-PTP depletion prevents multivesicular body sorting, whilst also blocking cargo from exiting Rab5-rich endosomes. HD-PTP depleted cells contain hyperphosphorylated Rabaptin-5, a cofactor for the Rab5 guanine nucleotide exchange factor, Rabex-5, though HD-PTP is unlikely to directly dephosphorylate Rabaptin-5. In addition, HD-PTP depleted cells exhibit Rabaptin-5 dependent hyperactivation of Rab5. HD-PTP binds directly to Rabaptin-5, between its Rabex-5 and Rab5 binding domains. This binding reaction involves the ESCRT-0/ESCRT-III binding site in HD-PTP and is competed by an ESCRT-III peptide. Jointly, these findings indicate that HD-PTP may alternately scaffold ESCRTs and modulate Rabex-5/Rabaptin-5 activity, thereby helping to coordinate the completion of MVB sorting with endosomal maturation.

TMCC3 localizes at the three-way junctions for the proper tubular network of the endoplasmic reticulum

2019 ◽  
Vol 476 (21) ◽  
pp. 3241-3260
Author(s):  
Sindhu Wisesa ◽  
Yasunori Yamamoto ◽  
Toshiaki Sakisaka
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Mammalian Cells ◽  
Coiled Coil ◽  
Transmembrane Domains ◽  
Domain Family ◽  
Coiled Coil Domain ◽  
U2os Cells ◽  
Er Membrane

The tubular network of the endoplasmic reticulum (ER) is formed by connecting ER tubules through three-way junctions. Two classes of the conserved ER membrane proteins, atlastins and lunapark, have been shown to reside at the three-way junctions so far and be involved in the generation and stabilization of the three-way junctions. In this study, we report TMCC3 (transmembrane and coiled-coil domain family 3), a member of the TEX28 family, as another ER membrane protein that resides at the three-way junctions in mammalian cells. When the TEX28 family members were transfected into U2OS cells, TMCC3 specifically localized at the three-way junctions in the peripheral ER. TMCC3 bound to atlastins through the C-terminal transmembrane domains. A TMCC3 mutant lacking the N-terminal coiled-coil domain abolished localization to the three-way junctions, suggesting that TMCC3 localized independently of binding to atlastins. TMCC3 knockdown caused a decrease in the number of three-way junctions and expansion of ER sheets, leading to a reduction of the tubular ER network in U2OS cells. The TMCC3 knockdown phenotype was partially rescued by the overexpression of atlastin-2, suggesting that TMCC3 knockdown would decrease the activity of atlastins. These results indicate that TMCC3 localizes at the three-way junctions for the proper tubular ER network.

scholarly journals A Novel Nanobody Precisely Visualizes Phosphorylated Histone H2AX in Living Cancer Cells under Drug-Induced Replication Stress

Cancers ◽  
2021 ◽  
Vol 13 (13) ◽  
pp. 3317
Author(s):  
Eric Moeglin ◽  
Dominique Desplancq ◽  
Audrey Stoessel ◽  
Christian Massute ◽  
Jeremy Ranniger ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Mammalian Cells ◽  
Chromatin Modification ◽  
Replication Stress ◽  
Living Cells ◽  
Single Step ◽  
Dna Double Strand Breaks ◽  
Drug Induced ◽  
Histone H2ax ◽  
C Terminus

Histone H2AX phosphorylated at serine 139 (γ-H2AX) is a hallmark of DNA damage, signaling the presence of DNA double-strand breaks and global replication stress in mammalian cells. While γ-H2AX can be visualized with antibodies in fixed cells, its detection in living cells was so far not possible. Here, we used immune libraries and phage display to isolate nanobodies that specifically bind to γ-H2AX. We solved the crystal structure of the most soluble nanobody in complex with the phosphopeptide corresponding to the C-terminus of γ-H2AX and show the atomic constituents behind its specificity. We engineered a bivalent version of this nanobody and show that bivalency is essential to quantitatively visualize γ-H2AX in fixed drug-treated cells. After labelling with a chemical fluorophore, we were able to detect γ-H2AX in a single-step assay with the same sensitivity as with validated antibodies. Moreover, we produced fluorescent nanobody-dTomato fusion proteins and applied a transduction strategy to visualize with precision γ-H2AX foci present in intact living cells following drug treatment. Together, this novel tool allows performing fast screenings of genotoxic drugs and enables to study the dynamics of this particular chromatin modification in individual cancer cells under a variety of conditions.

scholarly journals Membrane recruitment of Atg8 by Hfl1 facilitates turnover of vacuolar membrane proteins in yeast cells approaching stationary phase

BMC Biology ◽  
2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Cheng-Wen He ◽  
Xue-Fei Cui ◽  
Shao-Jie Ma ◽  
Qin Xu ◽  
Yan-Peng Ran ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Stationary Phase ◽  
Molecular Mechanisms ◽  
Multivesicular Body ◽  
Degradation Process ◽  
Vacuolar Membrane ◽  
Yeast Cells ◽  
Membrane Structures ◽  
Membrane Recruitment

Abstract Background The vacuole/lysosome is the final destination of autophagic pathways, but can also itself be degraded in whole or in part by selective macroautophagic or microautophagic processes. Diverse molecular mechanisms are involved in these processes, the characterization of which has lagged behind those of ATG-dependent macroautophagy and ESCRT-dependent endosomal multivesicular body pathways. Results Here we show that as yeast cells gradually exhaust available nutrients and approach stationary phase, multiple vacuolar integral membrane proteins with unrelated functions are degraded in the vacuolar lumen. This degradation depends on the ESCRT machinery, but does not strictly require ubiquitination of cargos or trafficking of cargos out of the vacuole. It is also temporally and mechanistically distinct from NPC-dependent microlipophagy. The turnover is facilitated by Atg8, an exception among autophagy proteins, and an Atg8-interacting vacuolar membrane protein, Hfl1. Lack of Atg8 or Hfl1 led to the accumulation of enlarged lumenal membrane structures in the vacuole. We further show that a key function of Hfl1 is the membrane recruitment of Atg8. In the presence of Hfl1, lipidation of Atg8 is not required for efficient cargo turnover. The need for Hfl1 can be partially bypassed by blocking Atg8 delipidation. Conclusions Our data reveal a vacuolar membrane protein degradation process with a unique dependence on vacuole-associated Atg8 downstream of ESCRTs, and we identify a specific role of Hfl1, a protein conserved from yeast to plants and animals, in membrane targeting of Atg8.

scholarly journals DNA-binding specificity of GATA family transcription factors.

1993 ◽  
Vol 13 (7) ◽  
pp. 3999-4010 ◽  
Author(s):  
M Merika ◽  
S H Orkin
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Mammalian Cells ◽  
Chimeric Protein ◽  
Binding Specificity ◽  
Mobility Shift ◽  
Binding Domains ◽  
Dna Binding Specificity ◽  
Mobility Shift Assay ◽  
Gata Family

GATA-binding proteins constitute a family of transcription factors that recognize a target site conforming to the consensus WGATAR (W = A or T and R = A or G). Here we have used the method of polymerase chain reaction-mediated random site selection to assess in an unbiased manner the DNA-binding specificity of GATA proteins. Contrary to our expectations, we show that GATA proteins bind a variety of motifs that deviate from the previously assigned consensus. Many of the nonconsensus sequences bind protein with high affinity, equivalent to that of conventional GATA motifs. By using the selected sequences as probes in the electrophoretic mobility shift assay, we demonstrate overlapping, but distinct, sequence preferences for GATA family members, specified by their respective DNA-binding domains. Furthermore, we provide additional evidence for interaction of amino and carboxy fingers of GATA-1 in defining its binding site. By performing cotransfection experiments, we also show that transactivation parallels DNA binding. A chimeric protein containing the finger domain of areA and the activation domains of GATA-1 is capable of activating transcription in mammalian cells through GATA motifs. Our findings suggest a mechanism by which GATA proteins might selectively regulate gene expression in cells in which they are coexpressed.

scholarly journals Staufen1 is imported into the nucleolus via a bipartite nuclear localization signal and several modulatory determinants

2005 ◽  
Vol 393 (1) ◽  
pp. 245-254 ◽  
Author(s):  
Catherine Martel ◽  
Paolo Macchi ◽  
Luc Furic ◽  
Michael A. Kiebler ◽  
Luc Desgroseillers
Keyword(s):  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Nuclear Export ◽  
Mammalian Cells ◽  
Rna Binding ◽  
Binding Activity ◽  
Nuclear Trafficking ◽  
Binding Domains ◽  
Bipartite Nuclear Localization Signal ◽  
Localization Signal

Mammalian Stau1 (Staufen1), a modular protein composed of several dsRBDs (double-stranded RNA-binding domains), is probably involved in mRNA localization. Although Stau1 is mostly described in association with the rough endoplasmic reticulum and ribosomes in the cytoplasm, recent studies suggest that it may transit through the nucleus/nucleolus. Using a sensitive yeast import assay, we show that Stau1 is actively imported into the nucleus through a newly identified bipartite nuclear localization signal. As in yeast, the bipartite nuclear localization signal is necessary for Stau1 nuclear import in mammalian cells. It is also required for Stau1 nucleolar trafficking. However, Stau1 nuclear transit seems to be regulated by mechanisms that involve cytoplasmic retention and/or facilitated nuclear export. Cytoplasmic retention is mainly achieved through the action of dsRBD3, with dsRBD2 playing a supporting role in this function. Similarly, dsRBD3, but not its RNA-binding activity, is critical for Stau1 nucleolar trafficking. The function of dsRBD3 is strengthened or stabilized by the presence of dsRBD4 but prevented by the interdomain between dsRBD2 and dsRBD3. Altogether, these results suggest that Stau1 nuclear trafficking is a highly regulated process involving several determinants. The presence of Stau1 in the nucleus/nucleolus suggests that it may be involved in ribonucleoprotein formation in the nucleus and/or in other nuclear functions not necessarily related to mRNA transport.

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