scholarly journals Membrane-anchored ubiquitin ligase complex is required for the turnover of lysosomal membrane proteins

2015 ◽  
Vol 211 (3) ◽  
pp. 639-652 ◽  
Author(s):  
Ming Li ◽  
Tatsuhiro Koshi ◽  
Scott D. Emr
Keyword(s):  
Membrane Proteins ◽  
Ubiquitin Ligase ◽  
Degradation Process ◽  
Degradation Pathway ◽  
Interacting Proteins ◽  
Vacuole Membrane ◽  
Ubiquitin Ligase Complex ◽  
Storage Organelle ◽  
Lysosomal Membrane Proteins ◽  
Nutrient Homeostasis

Cells must regulate the abundance and activity of numerous nutrient transporters in different organelle membranes to achieve nutrient homeostasis. As the recycling center and major storage organelle, lysosomes are essential for maintaining nutrient homeostasis. However, very little is known about mechanisms that govern the regulation of its membrane proteins. In this study, we demonstrated that changes of Zn2+ levels trigger the downregulation of vacuolar Zn2+ transporters. Low Zn2+ levels cause the degradation of the influx transporter Cot1, whereas high Zn2+ levels trigger the degradation of the efflux channel Zrt3. The degradation process depends on the vacuole membrane recycling and degradation pathway. Unexpectedly, we identified a RING domain–containing E3 ligase Tul1 and its interacting proteins in the Dsc complex that are important for the ubiquitination of Cot1 and partial ubiquitination of Zrt3. Our study demonstrated that the Dsc complex can function at the vacuole to regulate the composition and lifetime of vacuolar membrane proteins.

scholarly journals ESCRTs function directly on the lysosome membrane to downregulate ubiquitinated lysosomal membrane proteins

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Lu Zhu ◽  
Jeff R Jorgensen ◽  
Ming Li ◽  
Ya-Shan Chuang ◽  
Scott D Emr
Keyword(s):  
Membrane Proteins ◽  
Underlying Mechanism ◽  
Genetic Screens ◽  
Vacuole Membrane ◽  
Cargo Delivery ◽  
Ubiquitin Ligase Complex ◽  
Lysosomal Membrane Proteins ◽  
Homeostasis Regulation ◽  
Nutrient Homeostasis ◽  
Cargo Sorting

The lysosome plays an important role in maintaining cellular nutrient homeostasis. Regulation of nutrient storage can occur by the ubiquitination of certain transporters that are then sorted into the lysosome lumen for degradation. To better understand the underlying mechanism of this process, we performed genetic screens to identify components of the sorting machinery required for vacuole membrane protein degradation. These screens uncovered genes that encode a ubiquitin ligase complex, components of the PtdIns 3-kinase complex, and the ESCRT machinery. We developed a novel ubiquitination system, Rapamycin-Induced Degradation (RapiDeg), to test the sorting defects caused by these mutants. These tests revealed that ubiquitinated vacuole membrane proteins recruit ESCRTs to the vacuole surface, where they mediate cargo sorting and direct cargo delivery into the vacuole lumen. Our findings demonstrate that the ESCRTs can function at both the late endosome and the vacuole membrane to mediate cargo sorting and intra-luminal vesicle formation.

scholarly journals Sorting of a multi-subunit ubiquitin ligase complex in the endolysosome system

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Xi Yang ◽  
Felichi Mae Arines ◽  
Weichao Zhang ◽  
Ming Li
Keyword(s):  
Ubiquitin Ligase ◽  
Sequence Similarity ◽  
Degradation Pathway ◽  
Specific Protein ◽  
Biochemical Purification ◽  
Vacuole Membrane ◽  
Protein Levels ◽  
Ubiquitin Ligase Complex ◽  
Regulate Protein ◽  
Novel Model

The yeast Dsc E3 ligase complex has long been recognized as a Golgi-specific protein ubquitination system. It shares a striking sequence similarity to the Hrd1 complex that plays critical roles in the ER-associated degradation pathway. Using biochemical purification and mass spectrometry, we identified two novel Dsc subunits, which we named as Gld1 and Vld1. Surprisingly, Gld1 and Vld1 do not coexist in the same complex. Instead, they compete with each other to form two functionally independent Dsc subcomplexes. The Vld1 subcomplex takes the AP3 pathway to reach the vacuole membrane, whereas the Gld1 subcomplex travels through the VPS pathway and is cycled between Golgi and endosomes by the retromer. Thus, instead of being Golgi-specific, the Dsc complex can regulate protein levels at three distinct organelles, namely Golgi, endosome, and vacuole. Our study provides a novel model of achieving multi-tasking for transmembrane ubiquitin ligases with interchangeable trafficking adaptors.

scholarly journals A stalled retrotranslocation complex reveals physical linkage between substrate recognition and proteasomal degradation during ER-associated degradation

2013 ◽  
Vol 24 (11) ◽  
pp. 1765-1775 ◽  
Author(s):  
Kunio Nakatsukasa ◽  
Jeffrey L. Brodsky ◽  
Takumi Kamura
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Ubiquitin Ligase ◽  
Substrate Recognition ◽  
Proteasomal Degradation ◽  
26S Proteasome ◽  
Endoplasmic Reticulum Associated Degradation ◽  
Ubiquitin Ligase Complex ◽  
Physical Linkage ◽  
Tightly Coupled

During endoplasmic reticulum–associated degradation (ERAD), misfolded lumenal and membrane proteins in the ER are recognized by the transmembrane Hrd1 ubiquitin ligase complex and retrotranslocated to the cytosol for ubiquitination and degradation. Although substrates are believed to be delivered to the proteasome only after the ATPase Cdc48p/p97 acts, there is limited knowledge about how the Hrd1 complex coordinates with Cdc48p/p97 and the proteasome to orchestrate substrate recognition and degradation. Here we provide evidence that inactivation of Cdc48p/p97 stalls retrotranslocation and triggers formation of a complex that contains the 26S proteasome, Cdc48p/p97, ubiquitinated substrates, select components of the Hrd1 complex, and the lumenal recognition factor, Yos9p. We propose that the actions of Cdc48p/p97 and the proteasome are tightly coupled during ERAD. Our data also support a model in which the Hrd1 complex links substrate recognition and degradation on opposite sides of the ER membrane.

scholarly journals Quality Control of ER Membrane Proteins by the RNF185/Membralin Ubiquitin Ligase Complex

2020 ◽  
Vol 79 (5) ◽  
pp. 768-781.e7 ◽  
Author(s):  
Michael L. van de Weijer ◽  
Logesvaran Krshnan ◽  
Sabrina Liberatori ◽  
Elena Navarro Guerrero ◽  
Jacob Robson-Tull ◽  
...  
Keyword(s):  
Quality Control ◽  
Membrane Proteins ◽  
Ubiquitin Ligase ◽  
Ubiquitin Ligase Complex ◽  
Er Membrane

scholarly journals A Noncanonical Mechanism of Nrf2 Activation by Autophagy Deficiency: Direct Interaction between Keap1 and p62

2010 ◽  
Vol 30 (13) ◽  
pp. 3275-3285 ◽  
Author(s):  
Alexandria Lau ◽  
Xiao-Jun Wang ◽  
Fei Zhao ◽  
Nicole F. Villeneuve ◽  
Tongde Wu ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Defense Mechanism ◽  
Ectopic Expression ◽  
E3 Ubiquitin Ligase ◽  
Direct Interaction ◽  
Degradation Pathway ◽  
26S Proteasome ◽  
Ubiquitin Ligase Complex ◽  
Subsequent Degradation ◽  
Kelch Domain

ABSTRACT In response to stress, cells can utilize several cellular processes, such as autophagy, which is a bulk-lysosomal degradation pathway, to mitigate damages and increase the chances of cell survival. Deregulation of autophagy causes upregulation of p62 and the formation of p62-containing aggregates, which are associated with neurodegenerative diseases and cancer. The Nrf2-Keap1 pathway functions as a critical regulator of the cell's defense mechanism against oxidative stress by controlling the expression of many cellular protective proteins. Under basal conditions, Nrf2 is ubiquitinated by the Keap1-Cul3-E3 ubiquitin ligase complex and targeted to the 26S proteasome for degradation. Upon induction, the activity of the E3 ubiquitin ligase is inhibited through the modification of cysteine residues in Keap1, resulting in the stabilization and activation of Nrf2. In this current study, we identified the direct interaction between p62 and Keap1 and the residues required for the interaction have been mapped to 349-DPSTGE-354 in p62 and three arginines in the Kelch domain of Keap1. Accumulation of endogenous p62 or ectopic expression of p62 sequesters Keap1 into aggregates, resulting in the inhibition of Keap1-mediated Nrf2 ubiquitination and its subsequent degradation by the proteasome. In contrast, overexpression of mutated p62, which loses its ability to interact with Keap1, had no effect on Nrf2 stability, demonstrating that p62-mediated Nrf2 upregulation is Keap1 dependent. These findings demonstrate that autophagy deficiency activates the Nrf2 pathway in a noncanonical cysteine-independent mechanism.

scholarly journals A  conserved ubiquitin- and ESCRT-dependent pathway internalizes human lysosomal membrane proteins for degradation

PLoS Biology ◽  
2021 ◽  
Vol 19 (7) ◽  
pp. e3001361
Author(s):  
Weichao Zhang ◽  
Xi Yang ◽  
Liang Chen ◽  
Yun-Yu Liu ◽  
Varsha Venkatarangan ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Lysosomal Membrane ◽  
Dependent Manner ◽  
Endosomal Sorting ◽  
Escrt Machinery ◽  
Lysosomal Membrane Proteins ◽  
Carboxyl Terminal ◽  
Nutrient Homeostasis ◽  
Lysosome Membrane ◽  
Dependent Pathway

The lysosome is an essential organelle to recycle cellular materials and maintain nutrient homeostasis, but the mechanism to down-regulate its membrane proteins is poorly understood. In this study, we performed a cycloheximide (CHX) chase assay to measure the half-lives of approximately 30 human lysosomal membrane proteins (LMPs) and identified RNF152 and LAPTM4A as short-lived membrane proteins. The degradation of both proteins is ubiquitin dependent. RNF152 is a transmembrane E3 ligase that ubiquitinates itself, whereas LAPTM4A uses its carboxyl-terminal PY motifs to recruit NEDD4-1 for ubiquitination. After ubiquitination, they are internalized into the lysosome lumen by the endosomal sorting complexes required for transport (ESCRT) machinery for degradation. Strikingly, when ectopically expressed in budding yeast, human RNF152 is still degraded by the vacuole (yeast lysosome) in an ESCRT-dependent manner. Thus, our study uncovered a conserved mechanism to down-regulate lysosome membrane proteins.

scholarly journals A conserved ubiquitin- and ESCRT-dependent pathway to regulate human lysosomal membrane proteins

2020 ◽  
Author(s):  
Weichao Zhang ◽  
Xi Yang ◽  
Liang Chen ◽  
Yun-Yu Liu ◽  
Varsha Venkatarangan ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Cellular Materials ◽  
Lysosomal Membrane ◽  
Dependent Manner ◽  
Endosomal Sorting ◽  
Escrt Machinery ◽  
Lysosomal Membrane Proteins ◽  
Nutrient Homeostasis ◽  
Lysosome Membrane ◽  
Dependent Pathway

AbstractThe lysosome is an essential organelle to recycle cellular materials and maintain nutrient homeostasis, but the mechanism to down-regulate lysosomal membrane proteins is poorly understood. In this study, we developed a cycloheximide chase assay to measure the half-lives of ~30 human lysosomal membrane proteins, and identified RNF152 as a short-lived protein. The degradation of RNF152 depends on ubiquitin and the endosomal sorting complexes required for transport (ESCRT) machinery. Ubiquitinated RNF152 is sorted and internalized by the ESCRT machinery into the lysosomal lumen for degradation. Strikingly, when expressed in budding yeast, human RNF152 is also degraded by the vacuole (yeast lysosome) in an ESCRT-dependent manner. Thus, our study uncovered a conserved mechanism to down-regulate lysosome membrane proteins.

scholarly journals Quality Control of ER Membrane Proteins by the RNF185/Membralin Ubiquitin Ligase Complex

2020 ◽  
Vol 80 (2) ◽  
pp. 374-375
Author(s):  
Michael L. van de Weijer ◽  
Logesvaran Krshnan ◽  
Sabrina Liberatori ◽  
Elena Navarro Guerrero ◽  
Jacob Robson-Tull ◽  
...  
Keyword(s):  
Quality Control ◽  
Membrane Proteins ◽  
Ubiquitin Ligase ◽  
Ubiquitin Ligase Complex ◽  
Er Membrane

scholarly journals ESCRT, not intralumenal fragments, sorts ubiquitinated vacuole membrane proteins for degradation

2021 ◽  
Vol 220 (8) ◽  
Author(s):  
Xi Yang ◽  
Lucas Reist ◽  
Dominic A. Chomchai ◽  
Liang Chen ◽  
Felichi Mae Arines ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Degradation Process ◽  
Nutrient Sensing ◽  
Imaging Method ◽  
Membrane Composition ◽  
Vacuole Membrane ◽  
Internalization Mechanism ◽  
Vacuole Fusion ◽  
Competing Models

The lysosome (or vacuole in fungi and plants) is an essential organelle for nutrient sensing and cellular homeostasis. In response to environmental stresses such as starvation, the yeast vacuole can adjust its membrane composition by selectively internalizing membrane proteins into the lumen for degradation. Regarding the selective internalization mechanism, two competing models have been proposed. One model suggests that the ESCRT machinery is responsible for the sorting. In contrast, the ESCRT-independent intralumenal fragment (ILF) pathway proposes that the fragment generated by homotypic vacuole fusion is responsible for the sorting. Here, we applied a microfluidics-based imaging method to capture the complete degradation process in vivo. Combining live-cell imaging with a synchronized ubiquitination system, we demonstrated that ILF cargoes are not degraded through intralumenal fragments. Instead, ESCRTs function on the vacuole membrane to sort them into the lumen for degradation. We further discussed challenges in reconstituting vacuole membrane protein degradation.

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