scholarly journals The Bul1/2 Alpha-Arrestins Promote Ubiquitylation and Endocytosis of the Can1 Permease upon Cycloheximide-Induced TORC1-Hyperactivation

2021 ◽  
Vol 22 (19) ◽  
pp. 10208
Author(s):  
Amalia H. Megarioti ◽  
Cecilia Primo ◽  
George C. Kapetanakis ◽  
Alexandros Athanasopoulos ◽  
Vicky Sophianopoulou ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Ubiquitin Ligase ◽  
Molecular Mechanisms ◽  
Membrane Transporters ◽  
Environmental Cues ◽  
Specific Amino Acid ◽  
Major Mechanism ◽  
N Terminus ◽  
Golgi Network ◽  
Arginine Permease

Selective endocytosis followed by degradation is a major mechanism for downregulating plasma membrane transporters in response to specific environmental cues. In Saccharomyces cerevisiae, this endocytosis is promoted by ubiquitylation catalyzed by the Rsp5 ubiquitin-ligase, targeted to transporters via adaptors of the alpha-arrestin family. However, the molecular mechanisms of this targeting and their control according to conditions remain incompletely understood. In this work, we dissect the molecular mechanisms eliciting the endocytosis of Can1, the arginine permease, in response to cycloheximide-induced TORC1 hyperactivation. We show that cycloheximide promotes Rsp5-dependent Can1 ubiquitylation and endocytosis in a manner dependent on the Bul1/2 alpha-arrestins. Also crucial for this downregulation is a short acidic patch sequence in the N-terminus of Can1 likely acting as a binding site for Bul1/2. The previously reported inhibition by cycloheximide of transporter recycling, from the trans-Golgi network to the plasma membrane, seems to additionally contribute to efficient Can1 downregulation. Our results also indicate that, contrary to the previously described substrate-transport elicited Can1 endocytosis mediated by the Art1 alpha-arrestin, Bul1/2-mediated Can1 ubiquitylation occurs independently of the conformation of the transporter. This study provides further insights into how distinct alpha-arrestins control the ubiquitin-dependent downregulation of a specific amino acid transporter under different conditions.

scholarly journals Palmitoylation of Sindbis Virus TF Protein Regulates Its Plasma Membrane Localization and Subsequent Incorporation into Virions

2016 ◽  
Vol 91 (3) ◽  
Author(s):  
Jolene Ramsey ◽  
Emily C. Renzi ◽  
Randy J. Arnold ◽  
Jonathan C. Trinidad ◽  
Suchetana Mukhopadhyay
Keyword(s):  
Plasma Membrane ◽  
Sindbis Virus ◽  
Molecular Mechanisms ◽  
Wild Type Virus ◽  
Membrane Localization ◽  
Clear Understanding ◽  
Wild Type ◽  
Plasma Membrane Localization ◽  
C Terminus ◽  
N Terminus

ABSTRACT Palmitoylation is a reversible, posttranslational modification that helps target proteins to cellular membranes. The alphavirus small membrane proteins 6K and TF have been reported to be palmitoylated and to positively regulate budding. 6K and TF are isoforms that are identical in their N termini but unique in their C termini due to a −1 ribosomal frameshift during translation. In this study, we used cysteine (Cys) mutants to test differential palmitoylation of the Sindbis virus 6K and TF proteins. We modularly mutated the five Cys residues in the identical N termini of 6K and TF, the four additional Cys residues in TF's unique C terminus, or all nine Cys residues in TF. Using these mutants, we determined that TF palmitoylation occurs primarily in the N terminus. In contrast, 6K is not palmitoylated, even on these shared residues. In the C-terminal Cys mutant, TF protein levels increase both in the cell and in the released virion compared to the wild type. In viruses with the N-terminal Cys residues mutated, TF is much less efficiently localized to the plasma membrane, and it is not incorporated into the virion. The three Cys mutants have minor defects in cell culture growth but a high incidence of abnormal particle morphologies compared to the wild-type virus as determined by transmission electron microscopy. We propose a model where the C terminus of TF modulates the palmitoylation of TF at the N terminus, and palmitoylated TF is preferentially trafficked to the plasma membrane for virus budding. IMPORTANCE Alphaviruses are a reemerging viral cause of arthritogenic disease. Recently, the small 6K and TF proteins of alphaviruses were shown to contribute to virulence in vivo. Nevertheless, a clear understanding of the molecular mechanisms by which either protein acts to promote virus infection is missing. The TF protein is a component of budded virions, and optimal levels of TF correlate positively with wild-type-like particle morphology. In this study, we show that the palmitoylation of TF regulates its localization to the plasma membrane, which is the site of alphavirus budding. Mutants in which TF is not palmitoylated display drastically reduced plasma membrane localization, which effectively prevents TF from participating in budding or being incorporated into virus particles. Investigation of the regulation of TF will aid current efforts in the alphavirus field searching for approaches to mitigate alphaviral disease in humans.

scholarly journals Regulation of amino acid transporter trafficking by mTORC1 in primary human trophoblast cells is mediated by the ubiquitin ligase Nedd4-2

2016 ◽  
Vol 130 (7) ◽  
pp. 499-512 ◽  
Author(s):  
Fredrick J. Rosario ◽  
Kris Genelyn Dimasuay ◽  
Yoshikatsu Kanai ◽  
Theresa L. Powell ◽  
Thomas Jansson
Keyword(s):  
Plasma Membrane ◽  
Amino Acid ◽  
Ubiquitin Ligase ◽  
Membrane Trafficking ◽  
Molecular Mechanisms ◽  
Acid Transport ◽  
Trophoblast Cells ◽  
Human Trophoblast ◽  
Mtor Complex 1 ◽  
Human Trophoblast Cells

We demonstrate that mTOR complex 1 modulates amino acid transport in primary human trophoblast cells by regulating Nedd4-2 mediated ubiquitination and plasma membrane trafficking of specific transporter isoforms, which may constitute a molecular mechanisms underlying abnormal human fetal growth.

scholarly journals Role of N-glycosylation in trafficking of apical membrane proteins in epithelia

2009 ◽  
Vol 296 (3) ◽  
pp. F459-F469 ◽  
Author(s):  
Olga Vagin ◽  
Jeffrey A. Kraut ◽  
George Sachs
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Membrane Transporters ◽  
Membrane Domains ◽  
Sorting Signals ◽  
Galectin 3 ◽  
Vectorial Functions ◽  
Apical Sorting ◽  
Golgi Network

Polarized distribution of plasma membrane transporters and receptors in epithelia is essential for vectorial functions of epithelia. This polarity is maintained by sorting of membrane proteins into apical or basolateral transport containers in the trans-Golgi network and/or endosomes followed by their delivery to the appropriate plasma membrane domains. Sorting depends on the recognition of sorting signals in proteins by specific sorting machinery. In the present review, we summarize experimental evidence for and against the hypothesis that N-glycans attached to the membrane proteins can act as apical sorting signals. Furthermore, we discuss the roles of N-glycans in the apical sorting event per se and their contribution to folding and quality control of glycoproteins in the endoplasmic reticulum or retention of glycoproteins in the plasma membrane. Finally, we review existing hypotheses on the mechanism of apical sorting and discuss the potential roles of the lectins, VIP36 and galectin-3, as putative apical sorting receptors.

Mammary gland copper transport is stimulated by prolactin through alterations in Ctr1 and Atp7A localization

2006 ◽  
Vol 291 (4) ◽  
pp. R1181-R1191 ◽  
Author(s):  
Shannon L. Kelleher ◽  
Bo Lönnerdal
Keyword(s):  
Plasma Membrane ◽  
Mammary Gland ◽  
Molecular Mechanisms ◽  
Mammary Epithelial Cells ◽  
Milk Composition ◽  
Mammary Epithelial ◽  
Recycling Endosome ◽  
Protein Levels ◽  
Stage Of Lactation ◽  
Golgi Network

Milk copper (Cu) concentration declines and directly reflects the stage of lactation. Three Cu-specific transporters (Ctr1, Atp7A, Atp7B) have been identified in the mammary gland; however, the integrated role they play in milk Cu secretion is not understood. Whereas the regulation of milk composition by the lactogenic hormone prolactin (PRL) has been documented, the specific contribution of PRL to this process is largely unknown. Using the lactating rat as a model, we determined that the normal decline in milk Cu concentration parallels declining Cu availability to the mammary gland and is associated with decreased Atp7B protein levels. Mammary gland Cu transport was highest during early lactation and was stimulated by suckling and hyperprolactinemia, which was associated with Ctr1 and Atp7A localization at the plasma membrane. Using cultured mammary epithelial cells (HC11), we demonstrated that Ctr1 stains in association with intracellular vesicles that partially colocalize with transferrin receptor (recycling endosome marker). Atp7A was primarily colocalized with mannose 6-phosphate receptor (M6PR; late endosome marker), whereas Atp7B was partially colocalized with protein disulfide isomerase (endoplasmic reticulum marker), TGN38 ( trans-Golgi network marker) and M6PR. Prolactin stimulated Cu transport as a result of increased Ctr1 and Atp7A abundance at the plasma membrane. Although the molecular mechanisms responsible for these posttranslational changes are not understood, transient changes in prolactin signaling play a role in the regulation of mammary gland Cu secretion during lactation.

scholarly journals Cul3-KLHL20 E3 ubiquitin ligase plays a key role in the arms race between HIV-1 and host restriction

2021 ◽  
Author(s):  
Rongrong Li ◽  
Iqbal Ahmad ◽  
Sunan Li ◽  
Silas Johnson ◽  
Liangliang Sun ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Productive Infection ◽  
Restriction Factors ◽  
Host Restriction ◽  
Host Restriction Factor ◽  
Cullin 3 ◽  
Golgi Network ◽  
Hiv 1

Abstract HIV-1 must counteract various host restriction factors to establish productive infection. SERINC5 is a critical host restriction factor that potently blocks HIV-1 entry from virions, but its activity is counteracted by Nef. The SERINC5 and Nef activities are both initiated from the plasma membrane, where SERINC5 is packaged into virions and downregulated by Nef via lysosomal degradation. However, it is still unclear how SERINC5 is localized to the plasma membrane and how its expression is regulated on the plasma membrane. We now report that Cullin 3-KLHL20, a trans-Golgi network (TGN)-localized E3 ubiquitin ligase, polyubiquitinates SERINC5 at lysine 130 via K33- and K48-linked ubiquitin chains. The K130 polyubiquitination is required not only for the SERINC5 expression on the plasma membrane, but also the SERINC5 anti-HIV-1 activity and the Nef counteractive activity. Our study reveals an important role of K33/K48-branched ubiquitin chains in HIV-1 infection by regulating protein post-Golgi trafficking and degradation.

scholarly journals Dissociation of the intramolecularly cleaved N- and C-terminal fragments of the adhesion G protein-coupled receptor GPR133 (ADGRD1) increases canonical signaling

2020 ◽  
Author(s):  
Joshua D. Frenster ◽  
Gabriele Stephan ◽  
Niklas Ravn-Boess ◽  
Devin Bready ◽  
Jordan Wilcox ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
G Protein ◽  
Receptor Activation ◽  
List Type ◽  
G Protein Coupled Receptor ◽  
Major Mechanism ◽  
N Terminus ◽  
Adhesion Gpcr ◽  
G Protein Coupled

SUMMARYGPR133 (ADGRD1), an adhesion G protein-coupled receptor (GPCR), is necessary for growth of glioblastoma (GBM), a brain malignancy. The extracellular N-terminus of GPR133 is thought to be autoproteolytically cleaved into an N-terminal and a C-terminal fragment (NTF and CTF). Nevertheless, the role of this cleavage in receptor activation remains unclear. Here, we show that the wild-type (WT) receptor is cleaved after protein synthesis and generates significantly more canonical signaling than an uncleavable point mutant (H543R) in patient-derived GBM cultures and HEK293T cells. However, the resulting NTF and CTF remain non-covalently bound until the receptor is trafficked to the plasma membrane, where we find NTF-CTF dissociation. Using a fusion of the hPAR1 receptor N-terminus and the CTF of GPR133, we demonstrate that thrombin-induced cleavage and shedding of the hPAR1 NTF increases receptor signaling. This study supports a model where dissociation of the NTF at the plasma membrane promotes GPR133 activation.Highlights-GPR133 is intramolecularly cleaved in patient-derived GBM cultures-Cleaved GPR133 signals at higher efficacy than the uncleavable GPR133 H543R mutant-The N- and C-terminal fragments (NTF and CTF) of GPR133 dissociate at the plasma membrane-Acute thrombin-induced cleavage of the human PAR1 NTF from the GPR133 CTF increases signalingeTOC BlurbFrenster et al. demonstrate intramolecular cleavage of the adhesion GPCR GPR133 in glioblastoma and HEK293T cells. The resulting N- and C-terminal fragments dissociate at the plasma membrane to increase canonical signaling. The findings suggest dissociation of GPR133’s N-terminus at the plasma membrane represents a major mechanism of receptor activation.

Phosphoinositides and membrane traffic at the trans-Golgi network.

2005 ◽  
Vol 72 ◽  
pp. 31-38 ◽  
Author(s):  
Rawshan R. Choudhury ◽  
Noora Hyvola ◽  
Martin Lowe
Keyword(s):  
Plasma Membrane ◽  
Secretory Pathway ◽  
Binding Proteins ◽  
Molecular Mechanisms ◽  
Membrane Traffic ◽  
Trans Golgi Network ◽  
Cargo Proteins ◽  
Golgi Network

Cargo proteins moving along the secretory pathway are sorted at the TGN (trans-Golgi network) into distinct carriers for delivery to the plasma membrane or endosomes. Recent studies in yeast and mammals have shown that formation of these carriers is regulated by PtdIns(4)P. This phosphoinositide is abundant at the TGN and acts to recruit components required for carrier formation to the membrane. Other phosphoinositides are also present on the TGN, but the extent to which they regulate trafficking is less clear. Further characterization of phosphoinositide kinases and phosphatases together with identification of new TGN-associated phosphoinositide-binding proteins will reveal the extent to which different phosphoinositides regulate TGN trafficking, and help define the molecular mechanisms involved.

scholarly journals Molecular mechanisms of neurite extension

1999 ◽  
Vol 354 (1381) ◽  
pp. 387-394 ◽  
Author(s):  
Flavia Valtorta ◽  
Chiara Leoni
Keyword(s):  
Signal Transduction ◽  
Plasma Membrane ◽  
Recent Progress ◽  
Molecular Mechanisms ◽  
Environmental Cues ◽  
Major Task ◽  
Neurite Extension ◽  
Molecular Synthesis ◽  
Developing Neurons ◽  
Made In

The extension of neurites is a major task of developing neurons, requiring a significant metabolic effort to sustain the increase in molecular synthesis necessary for plasma membrane expansion. In addition, neurite extension involves changes in the subsets of expressed proteins and reorganization of the cytomatrix. These phenomena are driven by environmental cues which activate signal transduction processes as well as by the intrinsic genetic program of the cell. The present review summarizes some of the most recent progress made in the elucidation of the molecular mechanisms underlying these processes.

Downregulation of the broad-specificity amino acid permease Agp1 mediated by the ubiquitin ligase Rsp5 and the arrestin-like protein Bul1 in yeast

2021 ◽  
Vol 85 (5) ◽  
pp. 1266-1274
Author(s):  
Ryoya Tanahashi ◽  
Tomonori Matsushita ◽  
Akira Nishimura ◽  
Hiroshi Takagi
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Amino Acid ◽  
Ubiquitin Ligase ◽  
Genetic Screening ◽  
Biochemical Analysis ◽  
Adaptor Proteins ◽  
Membrane Transporters ◽  
Amino Acid Permease ◽  
Broad Specificity

ABSTRACT Most of plasma membrane transporters are downregulated by ubiquitination-dependent endocytosis to avoid the excess uptake of their substrates. In Saccharomyces cerevisiae, ubiquitination of transporters is mediated by the HECT-type ubiquitin ligase Rsp5. We report here a mechanism underlying the substrate-induced endocytosis of the broad-specificity amino acid permease Agp1. First, we found that Agp1 underwent ubiquitination and endocytosis in response to the addition of excess asparagine, which is a substrate of Agp1. Moreover, the substrate-induced internalization of Agp1 was dependent on the ubiquitination activity of Rsp5. Since Rsp5 requires α-arrestin family proteins as adaptors to bind with substrates, we next developed a method of genetic screening to identify adaptor proteins for Agp1 endocytosis. This screening and biochemical analysis revealed that Bul1, but not its paralogue Bul2, was essential for the substrate-induced endocytosis of Agp1. Our results support that the substrate-induced endocytosis of Agp1 requires Rsp5 and Bul1.

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