scholarly journals TORC1 regulates vacuole membrane composition through ubiquitin- and ESCRT-dependent microautophagy

2019 ◽  
Author(s):  
Xi Yang ◽  
Weichao Zhang ◽  
Xin Wen ◽  
Patrick J. Bulinski ◽  
Dominic A. Chomchai ◽  
...  
Keyword(s):  
Amino Acids ◽  
Membrane Proteins ◽  
Nutrient Limitation ◽  
Signaling Cascade ◽  
Complex Relationship ◽  
Membrane Composition ◽  
Cellular Adaptation ◽  
Vacuole Membrane ◽  
Lysosome Biogenesis ◽  
Vacuole Biogenesis

AbstractCellular adaptation in response to nutrient limitation requires the induction of autophagy and lysosome biogenesis for the efficient recycling of macromolecules. Here, we discovered that starvation and TORC1 inactivation not only lead to the upregulation of autophagy and vacuole proteins involved in recycling, but also result in the downregulation of many vacuole membrane proteins to supply amino acids as part of a vacuole remodeling process. Downregulation of vacuole membrane proteins is initiated by ubiquitination, which is accomplished by the coordination of multiple E3 ubiquitin ligases, including Rsp5, the Dsc complex, and a newly characterized E3 ligase, Pib1. The Dsc complex is negatively regulated by TORC1 through the Rim15-Ume6 signaling cascade. After ubiquitination, vacuole membrane proteins are sorted into the lumen for degradation by ESCRT-dependent microautophagy. Thus, our study uncovered a complex relationship between TORC1 inactivation and vacuole biogenesis.

scholarly journals TORC1 regulates vacuole membrane composition through ubiquitin- and ESCRT-dependent microautophagy

2020 ◽  
Vol 219 (3) ◽  
Author(s):  
Xi Yang ◽  
Weichao Zhang ◽  
Xin Wen ◽  
Patrick J. Bulinski ◽  
Dominic A. Chomchai ◽  
...  
Keyword(s):  
Amino Acids ◽  
Membrane Proteins ◽  
Signaling Cascade ◽  
Complex Relationship ◽  
Membrane Composition ◽  
Down Regulation ◽  
Cellular Adaptation ◽  
Vacuole Membrane ◽  
Lysosome Biogenesis ◽  
Vacuole Biogenesis

Cellular adaptation in response to nutrient limitation requires the induction of autophagy and lysosome biogenesis for the efficient recycling of macromolecules. Here, we discovered that starvation and TORC1 inactivation not only lead to the up-regulation of autophagy and vacuole proteins involved in recycling but also result in the down-regulation of many vacuole membrane proteins to supply amino acids as part of a vacuole remodeling process. Down-regulation of vacuole membrane proteins is initiated by ubiquitination, which is accomplished by the coordination of multiple E3 ubiquitin ligases, including Rsp5, the Dsc complex, and a newly characterized E3 ligase, Pib1. The Dsc complex is negatively regulated by TORC1 through the Rim15-Ume6 signaling cascade. After ubiquitination, vacuole membrane proteins are sorted into the lumen for degradation by ESCRT-dependent microautophagy. Thus, our study uncovered a complex relationship between TORC1 inactivation and vacuole biogenesis.

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.

scholarly journals Monitoring mitochondrial translation by pulse SILAC

2021 ◽  
Author(s):  
Koshi Imami ◽  
Matthias Selbach ◽  
Yasushi Ishihama
Keyword(s):  
Oxidative Stress ◽  
Amino Acids ◽  
Membrane Proteins ◽  
Translational Regulation ◽  
Isotope Labeling ◽  
Protein Complexes ◽  
Complex Iii ◽  
Mitochondrial Translation ◽  
Mitochondrial Ribosomes ◽  
Hydrophobic Nature

SummaryMitochondrial ribosomes are specialized to translate the 13 membrane proteins encoded in the mitochondrial genome, but it is challenging to quantify mitochondrial translation products due to their hydrophobic nature. Here, we introduce a proteomic method that combines biochemical isolation of mitochondria with pulse stable isotope labeling by amino acids in cell culture (pSILAC). Our method provides the highest protein coverage (quantifying 12 out of the 13 inner-membrane proteins; average 2-fold improvement over previous studies) with the shortest measurement time. We applied this method to uncover the global picture of (post)translational regulation of both mitochondrial- and nuclear-encoded proteins involved in the assembly of protein complexes that mediate oxidative phosphorylation (OXPHOS). The results allow us to infer the assembly order of complex components and/or partners, as exemplified by complex III. This method should be applicable to study mitochondrial translation programs in many contexts, including oxidative stress and mitochondrial disease.

scholarly journals A new family of integral membrane proteins involved in transport of aromatic amino acids in Escherichia coli.

1991 ◽  
Vol 173 (10) ◽  
pp. 3231-3234 ◽  
Author(s):  
J P Sarsero ◽  
P J Wookey ◽  
P Gollnick ◽  
C Yanofsky ◽  
A J Pittard
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Membrane Proteins ◽  
Aromatic Amino Acids ◽  
Integral Membrane Proteins ◽  
New Family

scholarly journals Negatively charged amino acids in the stalk region of membrane proteins reduce ectodomain shedding

2020 ◽  
Vol 295 (35) ◽  
pp. 12343-12352 ◽  
Author(s):  
Ryo Iwagishi ◽  
Rika Tanaka ◽  
Munenosuke Seto ◽  
Tomoyo Takagi ◽  
Naoko Norioka ◽  
...  
Keyword(s):  
Amino Acids ◽  
Membrane Proteins ◽  
Molecular Mechanisms ◽  
Alternative Exon ◽  
Ectodomain Shedding ◽  
Post Translational Modification ◽  
Charged Amino Acids ◽  
Juxtamembrane Region ◽  
Protein Variants ◽  
Variant Protein

Ectodomain shedding is a post-translational modification mechanism by which the entire extracellular domain of membrane proteins is liberated through juxtamembrane processing. Because shedding rapidly and irreversibly alters the characteristics of cells, this process is properly regulated. However, the molecular mechanisms governing the propensity of membrane proteins to shedding are largely unknown. Here, we present evidence that negatively charged amino acids within the stalk region, an unstructured juxtamembrane region at which shedding occurs, contribute to shedding susceptibility. We show that two activated leukocyte cell adhesion molecule (ALCAM) protein variants produced by alternative splicing have different susceptibilities to ADAM metallopeptidase domain 17 (ADAM17)-mediated shedding. Of note, the inclusion of a stalk region encoded by a 39-bp-long alternative exon conferred shedding resistance. We found that this alternative exon encodes a large proportion of negatively charged amino acids, which we demonstrate are indispensable for conferring the shedding resistance. We also show that the introduction of negatively charged amino acids into the stalk region of shedding-susceptible ALCAM variant protein attenuates its shedding. Furthermore, we observed that negatively charged amino acids residing in the stalk region of Erb-B2 receptor tyrosine kinase 4 (ERBB4) are indispensable for its shedding resistance. Collectively, our results indicate that negatively charged amino acids within the stalk region interfere with the shedding of multiple membrane proteins. We conclude that the composition of the stalk region determines the shedding susceptibility of membrane proteins.

scholarly journals Analysis of the interactions of sulfur-containing amino acids in membrane proteins

2016 ◽  
Vol 25 (8) ◽  
pp. 1517-1524 ◽  
Author(s):  
José C. Gómez-Tamayo ◽  
Arnau Cordomí ◽  
Mireia Olivella ◽  
Eduardo Mayol ◽  
Daniel Fourmy ◽  
...  
Keyword(s):  
Amino Acids ◽  
Membrane Proteins ◽  
Sulfur Containing

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 Recruitment and organization of ESCRT-0 and ubiquitinated cargo via condensation

2021 ◽  
Author(s):  
Sudeep Banjade ◽  
Lu Zhu ◽  
Jeffrey Jorgensen ◽  
Sho Suzuki ◽  
Scott D. Emr
Keyword(s):  
Membrane Proteins ◽  
Self Assembly ◽  
Cell Biology ◽  
Substrate Recognition ◽  
Initial Step ◽  
Binding Interactions ◽  
Vacuole Membrane ◽  
Multivalent Interactions ◽  
Cargo Sorting

AbstractThe general mechanisms by which ESCRTs are specifically recruited to various membranes, and how ESCRT subunits are spatially organized remain central questions in cell biology. At the endosome and lysosomes, ubiquitination of membrane proteins triggers ESCRT-mediated substrate recognition and degradation. Using the yeast lysosome/vacuole, we define the principles by which substrate engagement by ESCRTs occurs at this organelle. We find that multivalent interactions between ESCRT-0 and polyubiquitin is critical for substrate recognition at yeast vacuoles, with a lower-valency requirement for cargo engagement at endosomes. Direct recruitment of ESCRT-0 induces dynamic foci on the vacuole membrane, and forms fluid condensates in vitro with polyubiquitin. We propose that self-assembly of early ESCRTs induces condensation, an initial step in ESCRT-assembly/nucleation at membranes. This property can be tuned specifically at various organelles by modulating the number of binding interactions.One-Sentence SummaryCondensation of multivalent ESCRT-0/polyubiquitin assemblies organizes cargo sorting reactions at lysosomes

Sign in / Sign up

Export Citation Format

Share Document