scholarly journals Regulators of Vps4 ATPase Activity at Endosomes Differentially Influence the Size and Rate of Formation of Intralumenal Vesicles

2010 ◽  
Vol 21 (6) ◽  
pp. 1023-1032 ◽  
Author(s):  
Daniel P. Nickerson ◽  
Matthew West ◽  
Ryan Henry ◽  
Greg Odorizzi
Keyword(s):  
Subcellular Localization ◽  
Atpase Activity ◽  
Transmembrane Proteins ◽  
Regulatory Proteins ◽  
Multivesicular Bodies ◽  
Unique Role ◽  
Vesicle Size ◽  
Endosomal Sorting ◽  
Membrane Scission

Recruitment of endosomal sorting complexes required for transport (ESCRTs) to the cytosolic face of endosomes regulates selective inclusion of transmembrane proteins into the lumenal vesicles of multivesicular bodies (MVBs). ESCRT-0, -I, and -II bind directly to ubiquitinated transmembrane cargoes of the MVB pathway, whereas polymerization of ESCRT-III at endosomes is thought to bend the membrane and/or provide the energetic force that drives membrane scission and detachment of vesicles into the endosome lumen. Disassembly of the ESCRT-III polymer and dissociation of its subunits from endosomes requires the Vps4 ATPase, the activity of which is controlled in vivo by regulatory proteins. We identify distinct spatiotemporal roles for Vps4-regulating proteins through examinations of subcellular localization and endosome morphology. Did2 plays a unique role in the regulation of MVB lumenal vesicle size, whereas Vtal and Vps60 promote efficient membrane scission and delivery of membrane to the endosome lumen. These morphological effects probably result from Vps4-mediated manipulations of ESCRT-III, because we show dissociation of ESCRT-0, -I, and -II from endosomes is not directly dependent on Vps4 activity.

scholarly journals A conserved late endosome–targeting signal required for Doa4 deubiquitylating enzyme function

2006 ◽  
Vol 175 (5) ◽  
pp. 825-835 ◽  
Author(s):  
Alexander Amerik ◽  
Nazia Sindhi ◽  
Mark Hochstrasser
Keyword(s):  
Subcellular Localization ◽  
Terminal Segment ◽  
Late Endosome ◽  
Enzyme Specificity ◽  
Endosomal Sorting ◽  
Terminal Domain ◽  
Localization Signal ◽  
Targeting Signal ◽  
Processing Protease

Enzyme specificity in vivo is often controlled by subcellular localization. Yeast Doa4, a deubiquitylating enzyme (DUB), removes ubiquitin from membrane proteins destined for vacuolar degradation. Doa4 is recruited to the late endosome after ESCRT-III (endosomal sorting complex required for transport III) has assembled there. We show that an N-terminal segment of Doa4 is sufficient for endosome association. This domain bears four conserved elements (boxes A–D). Deletion of the most conserved of these, A or B, prevents Doa4 endosomal localization. These mutants cannot sustain ubiquitin-dependent proteolysis even though neither motif is essential for deubiquitylating activity. Ubiquitin-specific processing protease 5 (Ubp5), the closest paralogue of Doa4, has no functional overlap. Ubp5 concentrates at the bud neck; its N-terminal domain is critical for this. Importantly, substitution of the Ubp5 N-terminal domain with that of Doa4 relocalizes the Ubp5 enzyme to endosomes and provides Doa4 function. This is the first demonstration of a physiologically important DUB subcellular localization signal and provides a striking example of the functional diversification of DUB paralogues by the evolution of alternative spatial signals.

scholarly journals GCUNC-45 Is a Novel Regulator for the Progesterone Receptor/hsp90 Chaperoning Pathway

2006 ◽  
Vol 26 (5) ◽  
pp. 1722-1730 ◽  
Author(s):  
Ahmed Chadli ◽  
J. Dinny Graham ◽  
M. Greg Abel ◽  
Twila A. Jackson ◽  
David F. Gordon ◽  
...  
Keyword(s):  
Progesterone Receptor ◽  
Atpase Activity ◽  
Binding Site ◽  
Signaling Pathway ◽  
Regulatory Proteins ◽  
N Terminus ◽  
Positive Factor

ABSTRACT The hsp90 chaperoning pathway is a multiprotein system that is required for the production or activation of many cell regulatory proteins, including the progesterone receptor (PR). We report here the identity of GCUNC-45 as a novel modulator of PR chaperoning by hsp90. GCUNC-45, previously implicated in the activities of myosins, can interact in vivo and in vitro with both PR-A and PR-B and with hsp90. Overexpression and knockdown experiments show GCUNC-45 to be a positive factor in promoting PR function in the cell. GCUNC-45 binds to the ATP-binding domain of hsp90 to prevent the activation of its ATPase activity by the cochaperone Aha1. This effect limits PR chaperoning by hsp90, but this can be reversed by FKBP52, a cochaperone that is thought to act later in the pathway. These findings reveal a new cochaperone binding site near the N terminus of hsp90, add insight on the role of FKBP52, and identify GCUNC-45 as a novel regulator of the PR signaling pathway.

scholarly journals Efficient Cargo Sorting by ESCRT-I and the Subsequent Release of ESCRT-I from Multivesicular Bodies Requires the Subunit Mvb12

2007 ◽  
Vol 18 (2) ◽  
pp. 636-645 ◽  
Author(s):  
Matt Curtiss ◽  
Charles Jones ◽  
Markus Babst
Keyword(s):  
Protein Complex ◽  
Transmembrane Proteins ◽  
Multivesicular Body ◽  
Multivesicular Bodies ◽  
Rapid Degradation ◽  
Endosomal Sorting ◽  
Escrt Machinery ◽  
Complex Functions ◽  
Vacuolar Protein ◽  
Cargo Sorting

The endosomal sorting complex required for transport (ESCRT)-I protein complex functions in recognition and sorting of ubiquitinated transmembrane proteins into multivesicular body (MVB) vesicles. It has been shown that ESCRT-I contains the vacuolar protein sorting (Vps) proteins Vps23, Vps28, and Vps37. We identified an additional subunit of yeast ESCRT-I called Mvb12, which seems to associate with ESCRT-I by binding to Vps37. Transient recruitment of ESCRT-I to MVBs results in the rapid degradation of Mvb12. In contrast to mutations in other ESCRT-I subunits, which result in strong defects in MVB cargo sorting, deletion of MVB12 resulted in only a partial sorting phenotype. This trafficking defect was fully suppressed by overexpression of the ESCRT-II complex. Mutations in MVB12 did not affect recruitment of ESCRT-I to MVBs, but they did result in delivery of ESCRT-I to the vacuolar lumen via the MVB pathway. Together, these observations suggest that Mvb12 may function in regulating the interactions of ESCRT-I with cargo and other proteins of the ESCRT machinery to efficiently coordinate cargo sorting and release of ESCRT-I from the MVB.

scholarly journals Bro1 stimulates Vps4 to promote intralumenal vesicle formation during multivesicular body biogenesis

2021 ◽  
Vol 220 (8) ◽  
Author(s):  
Chun-Che Tseng ◽  
Shirley Dean ◽  
Brian A. Davies ◽  
Ishara F. Azmi ◽  
Natalya Pashkova ◽  
...  
Keyword(s):  
Multivesicular Body ◽  
Vesicle Formation ◽  
Multivesicular Bodies ◽  
Membrane Remodeling ◽  
Endosomal Sorting ◽  
Aaa Atpase ◽  
Ubiquitin Binding ◽  
Stimulation Of ◽  
Cargo Sorting

Endosomal sorting complexes required for transport (ESCRT-0, -I, -II, -III) execute cargo sorting and intralumenal vesicle (ILV) formation during conversion of endosomes to multivesicular bodies (MVBs). The AAA-ATPase Vps4 regulates the ESCRT-III polymer to facilitate membrane remodeling and ILV scission during MVB biogenesis. Here, we show that the conserved V domain of ESCRT-associated protein Bro1 (the yeast homologue of mammalian proteins ALIX and HD-PTP) directly stimulates Vps4. This activity is required for MVB cargo sorting. Furthermore, the Bro1 V domain alone supports Vps4/ESCRT–driven ILV formation in vivo without efficient MVB cargo sorting. These results reveal a novel activity of the V domains of Bro1 homologues in licensing ESCRT-III–dependent ILV formation and suggest a role in coordinating cargo sorting with membrane remodeling during MVB sorting. Moreover, ubiquitin binding enhances V domain stimulation of Vps4 to promote ILV formation via the Bro1–Vps4–ESCRT-III axis, uncovering a novel role for ubiquitin during MVB biogenesis in addition to facilitating cargo recognition.

scholarly journals Bro1 binding to Snf7 regulates ESCRT-III membrane scission activity in yeast

2011 ◽  
Vol 192 (2) ◽  
pp. 295-306 ◽  
Author(s):  
Megan Wemmer ◽  
Ishara Azmi ◽  
Matthew West ◽  
Brian Davies ◽  
David Katzmann ◽  
...  
Keyword(s):  
Electron Tomography ◽  
Enveloped Viruses ◽  
Endosomal Sorting ◽  
Reduced Frequency ◽  
Stabilization Effect ◽  
Auxiliary Protein ◽  
Membrane Scission ◽  
The Stability

Endosomal sorting complexes required for transport (ESCRTs) promote the invagination of vesicles into the lumen of endosomes, the budding of enveloped viruses, and the separation of cells during cytokinesis. These processes share a topologically similar membrane scission event facilitated by ESCRT-III assembly at the cytosolic surface of the membrane. The Snf7 subunit of ESCRT-III in yeast binds directly to an auxiliary protein, Bro1. Like ESCRT-III, Bro1 is required for the formation of intralumenal vesicles at endosomes, but its role in membrane scission is unknown. We show that overexpression of Bro1 or its N-terminal Bro1 domain that binds Snf7 enhances the stability of ESCRT-III by inhibiting Vps4-mediated disassembly in vivo and in vitro. This stabilization effect correlates with a reduced frequency in the detachment of intralumenal vesicles as observed by electron tomography, implicating Bro1 as a regulator of ESCRT-III disassembly and membrane scission activity.

scholarly journals Coordinated binding of Vps4 to ESCRT-III drives membrane neck constriction during MVB vesicle formation

2014 ◽  
Vol 205 (1) ◽  
pp. 33-49 ◽  
Author(s):  
Manuel Alonso Y Adell ◽  
Georg F. Vogel ◽  
Mehrshad Pakdel ◽  
Martin Müller ◽  
Herbert Lindner ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Adenosine Triphosphatase ◽  
Electron Tomography ◽  
Vesicle Formation ◽  
Multivesicular Bodies ◽  
Membrane Remodeling ◽  
Yeast Genetics ◽  
Endosomal Sorting ◽  
Distinct Membrane

Five endosomal sorting complexes required for transport (ESCRTs) mediate the degradation of ubiquitinated membrane proteins via multivesicular bodies (MVBs) in lysosomes. ESCRT-0, -I, and –II interact with cargo on endosomes. ESCRT-II also initiates the assembly of a ringlike ESCRT-III filament consisting of Vps20, Snf7, Vps24, and Vps2. The AAA–adenosine triphosphatase Vps4 disassembles and recycles the ESCRT-III complex, thereby terminating the ESCRT pathway. A mechanistic role for Vps4 in intraluminal vesicle (ILV) formation has been unclear. By combining yeast genetics, biochemistry, and electron tomography, we find that ESCRT-III assembly on endosomes is required to induce or stabilize the necks of growing MVB ILVs. Yet, ESCRT-III alone is not sufficient to complete ILV biogenesis. Rather, binding of Vps4 to ESCRT-III, coordinated by interactions with Vps2 and Snf7, is coupled to membrane neck constriction during ILV formation. Thus, Vps4 not only recycles ESCRT-III subunits but also cooperates with ESCRT-III to drive distinct membrane-remodeling steps, which lead to efficient membrane scission at the end of ILV biogenesis in vivo.

scholarly journals Interactions of ubiquitin and CHMP5 with the V domain of HD-PTP reveals role for regulation of Vps4 ATPase

2021 ◽  
Author(s):  
Natalya Pashkova ◽  
Liping Yu ◽  
Nicholas J. Schnicker ◽  
Chun-Che Tseng ◽  
Lokesh Gakhar ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Molecular Mechanism ◽  
Structural Basis ◽  
Multivesicular Bodies ◽  
Membrane Remodeling ◽  
Endosomal Sorting ◽  
Aaa Atpase ◽  
Ubiquitinated Proteins ◽  
The Family ◽  
Ubiquitin Binding

The family of Bro1 proteins coordinates the activity of the Endosomal Sorting Complexes Required for Transport (ESCRTs) to mediate a number of membrane remodeling events. These events culminate in membrane scission catalyzed by ESCRT-III, whose polymerization and disassembly is controlled by the AAA-ATPase, Vps4. Bro1-family members Alix and HD-PTP as well as yeast Bro1 have a central ‘V’ domains that non-covalently bind Ub and connect ubiquitinated proteins to ESCRT-driven functions such as the incorporation of ubiquitinated membrane proteins into intralumenal vesicles of multivesicular bodies. Recently, it was discovered that the V domain of yeast Bro1 binds the MIT domain of Vps4 to stimulate its ATPase activity. Here we determine the structural basis for how the V domain of human HD-PTP binds ubiquitin. The HD-PTP V domain also binds the MIT domain of Vps4 and ubiquitin-binding to the HD-PTP V domain enhances its ability to stimulate Vps4 ATPase activity. Additionally, we found V domains of both HD-PTP and Bro1 bind CHMP5 and Vps60, respectively, providing another potential molecular mechanism to alter Vps4 activity. These data support a model whereby contacts between ubiquitin, ESCRT-III, and Vps4 by V domains of the Bro1 family may coordinate late events in ESCRT-driven membrane remodeling events.

Meiotic CENP-C is a shepherd: bridging the space between the centromere and the kinetochore in time and space

2020 ◽  
Vol 64 (2) ◽  
pp. 251-261
Author(s):  
Jessica E. Fellmeth ◽  
Kim S. McKim
Keyword(s):  
Chromosome Segregation ◽  
New Technologies ◽  
Early Prophase ◽  
Unique Role ◽  
Kinetochore Assembly ◽  
M Phase ◽  
In Vivo Analysis ◽  
Meiosis I

Abstract While many of the proteins involved in the mitotic centromere and kinetochore are conserved in meiosis, they often gain a novel function due to the unique needs of homolog segregation during meiosis I (MI). CENP-C is a critical component of the centromere for kinetochore assembly in mitosis. Recent work, however, has highlighted the unique features of meiotic CENP-C. Centromere establishment and stability require CENP-C loading at the centromere for CENP-A function. Pre-meiotic loading of proteins necessary for homolog recombination as well as cohesion also rely on CENP-C, as do the main scaffolding components of the kinetochore. Much of this work relies on new technologies that enable in vivo analysis of meiosis like never before. Here, we strive to highlight the unique role of this highly conserved centromere protein that loads on to centromeres prior to M-phase onset, but continues to perform critical functions through chromosome segregation. CENP-C is not merely a structural link between the centromere and the kinetochore, but also a functional one joining the processes of early prophase homolog synapsis to late metaphase kinetochore assembly and signaling.

scholarly journals Subcellular Localization and Vesicular Structures of Anthocyanin Pigmentation by Fluorescence Imaging of Black Rice (Oryza sativa L.) Stigma Protoplast

Plants ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 685
Author(s):  
Enerand Mackon ◽  
Yafei Ma ◽  
Guibeline Charlie Jeazet Dongho Epse Mackon ◽  
Qiufeng Li ◽  
Qiong Zhou ◽  
...  
Keyword(s):  
Oryza Sativa ◽  
Subcellular Localization ◽  
Oryza Sativa L ◽  
Scientific Evidence ◽  
Brown Rice ◽  
The Body ◽  
Central Vacuole ◽  
In Planta ◽  
Black Rice

Anthocyanins belong to the group of flavonoid compounds broadly distributed in plant species responsible for attractive colors. In black rice (Oryza sativa L.), they are present in the stems, leaves, stigmas, and caryopsis. However, there is still no scientific evidence supporting the existence of compartmentalization and trafficking of anthocyanin inside the cells. In the current study, we took advantage of autofluorescence with anthocyanin’s unique excitation/emission properties to elucidate the subcellular localization of anthocyanin and report on the in planta characterization of anthocyanin prevacuolar vesicles (APV) and anthocyanic vacuolar inclusion (AVI) structure. Protoplasts were isolated from the stigma of black and brown rice and imaging using a confocal microscope. Our result showed the fluorescence displaying magenta color in purple stigma and no fluorescence in white stigma when excitation was provided by a helium–neon 552 nm and emission long pass 610–670 nm laser. The fluorescence was distributed throughout the cell, mainly in the central vacuole. Fluorescent images revealed two pools of anthocyanin inside the cells. The diffuse pools were largely found inside the vacuole lumen, while the body structures could be observed mostly inside the cytoplasm (APV) and slightly inside the vacuole (AVI) with different shapes, sizes, and color intensity. Based on their sizes, AVI could be grouped into small (Ф < 0.5 um), middle (Ф between 0.5 and 1 um), and large size (Ф > 1 um). Together, these results provided evidence about the sequestration and trafficking of anthocyanin from the cytoplasm to the central vacuole and the existence of different transport mechanisms of anthocyanin. Our results suggest that stigma cells are an excellent system for in vivo studying of anthocyanin in rice and provide a good foundation for understanding anthocyanin metabolism in plants, sequestration, and trafficking in black rice.

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