scholarly journals Calcineurin-dependent regulation of endocytosis by a plasma membrane ubiquitin ligase adaptor, Rcr1

2020 ◽  
Vol 219 (8) ◽  
Author(s):  
Lu Zhu ◽  
Richa Sardana ◽  
Daniel K. Jin ◽  
Scott D. Emr
Keyword(s):  
Plasma Membrane ◽  
Ubiquitin Ligase ◽  
Adaptor Proteins ◽  
Cell Integrity ◽  
Calcium Treatment ◽  
Cellular Processes ◽  
Acute Response ◽  
Exogenous Calcium ◽  
Sorting Motif ◽  
Py Motif

Rsp5, the Nedd4 family member in yeast, is an E3 ubiquitin ligase involved in numerous cellular processes, many of which require Rsp5 to interact with PY-motif containing adaptor proteins. Here, we show that two paralogous transmembrane Rsp5 adaptors, Rcr1 and Rcr2, are sorted to distinct cellular locations: Rcr1 is a plasma membrane (PM) protein, whereas Rcr2 is sorted to the vacuole. Rcr2 is delivered to the vacuole using ubiquitin as a sorting signal. Rcr1 is delivered to the PM by the exomer complex using a newly uncovered PM sorting motif. Further, we show that Rcr1, but not Rcr2, is up-regulated via the calcineurin/Crz1 signaling pathway. Upon exogenous calcium treatment, Rcr1 ubiquitinates and down-regulates the chitin synthase Chs3. We propose that the PM-anchored Rsp5/Rcr1 ubiquitin ligase-adaptor complex can provide an acute response to degrade unwanted proteins under stress conditions, thereby maintaining cell integrity.

scholarly journals Endocytosis of the Aspartic Acid/Glutamic Acid Transporter Dip5 Is Triggered by Substrate-Dependent Recruitment of the Rsp5 Ubiquitin Ligase via the Arrestin-Like Protein Aly2

2010 ◽  
Vol 30 (24) ◽  
pp. 5598-5607 ◽  
Author(s):  
Riko Hatakeyama ◽  
Masao Kamiya ◽  
Terunao Takahara ◽  
Tatsuya Maeda
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Glutamic Acid ◽  
Aspartic Acid ◽  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Acid Transporter ◽  
Py Motif ◽  
Related Proteins

ABSTRACT Endocytosis of nutrient transporters is stimulated under various conditions, such as elevated nutrient availability. In Saccharomyces cerevisiae, endocytosis is triggered by ubiquitination of transporters catalyzed by the E3 ubiquitin ligase Rsp5. However, how the ubiquitination is accelerated under certain conditions remains obscure. Here we demonstrate that closely related proteins Aly2/Art3 and Aly1/Art6, which are poorly characterized members of the arrestin-like protein family, mediate endocytosis of the aspartic acid/glutamic acid transporter Dip5. In aly2Δ cells, Dip5 is stabilized at the plasma membrane and is not endocytosed efficiently. Efficient ubiquitination of Dip5 is dependent on Aly2. aly1Δ cells also show deficiency in Dip5 endocytosis, although less remarkably than aly2Δ cells. Aly2 physically interacts in vivo with Rsp5 at its PY motif and also with Dip5, thus serving as an adaptor linking Rsp5 with Dip5 to achieve Dip5 ubiquitination. Importantly, the interaction between Aly2 and Dip5 is accelerated in response to elevated aspartic acid availability. This result indicates that the regulation of Dip5 endocytosis is accomplished by dynamic recruitment of Rsp5 via Aly2.

scholarly journals Adaptor linked K63 di-Ubiquitin activates Nedd4/Rsp5 E3 ligase

2021 ◽  
Author(s):  
Lu Zhu ◽  
Qing Zhang ◽  
Ciro Cordeiro ◽  
Sudeep Banjade ◽  
Richa Sardana ◽  
...  
Keyword(s):  
E3 Ligase ◽  
Adaptor Proteins ◽  
Regulation Mechanism ◽  
Hect Domain ◽  
E3 Ligases ◽  
Cellular Processes ◽  
Protein Ubiquitination ◽  
Cargo Protein ◽  
Py Motif ◽  
Substrate Induction

Nedd4/Rsp5 family E3 ligases mediate numerous cellular processes, many of which require the E3 ligase to interact with PY-motif containing adaptor proteins. Several Arrestin-Related Trafficking adaptors(ARTs) of Rsp5 were self-ubiquitinated for activation, but the regulation mechanism remains elusive. Remarkably, we demonstrate that Art1, Art4, and Art5 undergo K63-linked di-ubiquitination by Rsp5. This modification enhances the PM recruitment of Rsp5 by Art1 or Art5 upon substrate induction, required for cargo protein ubiquitination. In agreement with these observations, we find that di-ubiquitin strengthens the interaction between the Pombe orthologs of Rsp5 and Art1, Pub1 and Any1. Further, we discover that the HECT domain exosite protects the K63-linked di-ubiquitin on the adaptors from cleavage by the deubiquitination enzyme Ubp2. Strikingly, loss of this protection results in the loss of K63-linked di-ubiquitin from the adaptors and diverts the adaptors for K48-linked poly-ubiquitination and proteasome-mediated degradation. Together, our study uncovers a novel ubiquitination modification implemented by Rsp5 adaptor proteins, underscoring the regulatory mechanism of how adaptor proteins control the recruitment and activity of Rsp5 for the turnover of membrane proteins.  

Versatile role of the yeast ubiquitin ligase Rsp5p in intracellular trafficking

2008 ◽  
Vol 36 (5) ◽  
pp. 791-796 ◽  
Author(s):  
Naima Belgareh-Touzé ◽  
Sébastien Léon ◽  
Zoi Erpapazoglou ◽  
Marta Stawiecka-Mirota ◽  
Danièle Urban-Grimal ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Ubiquitin Ligase ◽  
Protein Trafficking ◽  
Adaptor Proteins ◽  
Binding Domains ◽  
Independent Functions ◽  
Membrane Protein Trafficking ◽  
Ubiquitin Ligase E3

The ubiquitin ligase (E3) Rsp5p is the only member of the Nedd (neural-precursor-cell-expressed, developmentally down-regulated) 4 family of E3s present in yeast. Rsp5p has several proteasome-independent functions in membrane protein trafficking, including a role in the ubiquitination of most plasma membrane proteins, leading to their endocytosis. Rsp5p is also required for the ubiquitination of endosomal proteins, leading to their sorting to the internal vesicles of MVBs (multivesicular bodies). Rsp5p catalyses the attachment of non-conventional ubiquitin chains, linked through ubiquitin Lys-63, to some endocytic and MVB cargoes. This modification appears to be required for efficient sorting, possibly because these chains have a greater affinity for the ubiquitin-binding domains present within endocytic or MVB sorting complexes. The mechanisms involved in the recognition of plasma membrane and MVB substrates by Rsp5p remain unclear. A subset of Rsp5/Nedd4 substrates have a ‘PY motif’ and are recognized directly by the WW (Trp-Trp) domains of Rsp5p. Most Rsp5p substrates do not carry PY motifs, but some may depend on PY-containing proteins for their ubiquitination by Rsp5p, consistent with the latter's acting as specificity factors or adaptors. As in other ubiquitin-conjugating systems, these adaptors are also Rsp5p substrates and undergo ubiquitin-dependent trafficking. In the present review, we discuss recent examples illustrating the role of Rsp5p in membrane protein trafficking and providing new insights into the regulation of this E3 by adaptor proteins.

scholarly journals Syp1 regulates the clathrin-mediated and clathrin-independent endocytosis of multiple cargo proteins through a novel sorting motif

2017 ◽  
Vol 28 (18) ◽  
pp. 2434-2448 ◽  
Author(s):  
Amanda Reider Apel ◽  
Kyle Hoban ◽  
Silvia Chuartzman ◽  
Raffi Tonikian ◽  
Sachdev Sidhu ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Phage Display ◽  
Nutrient Uptake ◽  
Surface Composition ◽  
Adaptor Proteins ◽  
Endocytic Pathway ◽  
Peptide Transporter ◽  
Cargo Proteins ◽  
Sorting Motif

Internalization of proteins from the plasma membrane (PM) allows for cell-surface composition regulation, signaling of network modulation, and nutrient uptake. Clathrin-mediated endocytosis (CME) is a major internalization route for PM proteins. During CME, endocytic adaptor proteins bind cargoes at the cell surface and link them to the PM and clathrin coat. Muniscins are a conserved family of endocytic adaptors, including Syp1 in budding yeast and its mammalian orthologue, FCHo1. These adaptors bind cargo via a C-terminal μ-homology domain (μHD); however, few cargoes exhibiting muniscin-dependent endocytosis have been identified, and the sorting sequence recognized by the µHD is unknown. To reveal Syp1 cargo-sorting motifs, we performed a phage display screen and used biochemical methods to demonstrate that the Syp1 µHD binds DxY motifs in the previously identified Syp1 cargo Mid2 and the v-SNARE Snc1. We also executed an unbiased visual screen, which identified the peptide transporter Ptr2 and the ammonium permease Mep3 as Syp1 cargoes containing DxY motifs. Finally, we determined that, in addition to regulating cargo entry through CME, Syp1 can promote internalization of Ptr2 through a recently identified clathrin-independent endocytic pathway that requires the Rho1 GTPase. These findings elucidate the mechanism of Syp1 cargo recognition and its role in trafficking.

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.

vitreal cells and specialized phagocytes in the chick embryo retina: A TEM and SEM study

1990 ◽  
Vol 48 (3) ◽  
pp. 330-331
Author(s):  
M.A. Cuadros ◽  
M.J. Martinez-Guerrero ◽  
A. Rios
Keyword(s):  
Cell Death ◽  
Plasma Membrane ◽  
Acid Phosphatase ◽  
Chick Embryo ◽  
Basement Membrane ◽  
Sem Images ◽  
Intimate Contact ◽  
Cellular Processes ◽  
Sem Study ◽  
Free Cells

In the chick embryo retina (days 3-4 of incubation), coinciding with an increase in cell death, specialized phagocytes characterized by intense acid phosphatase activity have been described. In these preparations, all free cells in the vitreal humor (vitreal cells) were strongly labeled. Conventional TEM and SEM techniques were used to characterize them and attempt to determine their relationship with retinal phagocytes.Two types of vitreal cells were distinguished. The first are located at some distance from the basement membrane of the neuroepithelium, and are rounded, with numerous vacuoles and thin cytoplasmic prolongations. Images of exo- and or endocytosis were frequent; the cells showed a well-developed Golgi apparatus (Fig. 1) In SEM images, the cells was covered with short cellular processes (Fig. 3). Cells lying parallel to or alongside the basement membrane are elongated. The plasma membrane is frequently in intimate contact with the basement membrane. These cells have generally a large cytoplasmic expansion (Fig. 5).

Saccharomyces cerevisiae SSD1-VConfers Longevity by a Sir2p-Independent Mechanism

Genetics ◽  
2004 ◽  
Vol 166 (4) ◽  
pp. 1661-1672 ◽  
Author(s):  
Matt Kaeberlein ◽  
Alex A Andalis ◽  
Gregory B Liszt ◽  
Gerald R Fink ◽  
Leonard Guarente
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Life Span ◽  
Cell Cycle Progression ◽  
Polymorphic Locus ◽  
Cell Integrity ◽  
Cycle Progression ◽  
Cellular Processes ◽  
Maximal Life Span ◽  
Independent Mechanism ◽  
Yeast Aging

AbstractThe SSD1 gene of Saccharomyces cerevisiae is a polymorphic locus that affects diverse cellular processes including cell integrity, cell cycle progression, and growth at high temperature. We show here that the SSD1-V allele is necessary for cells to achieve extremely long life span. Furthermore, addition of SSD1-V to cells can increase longevity independently of SIR2, although SIR2 is necessary for SSD1-V cells to attain maximal life span. Past studies of yeast aging have been performed in short-lived ssd1-d strain backgrounds. We propose that SSD1-V defines a previously undescribed pathway affecting cellular longevity and suggest that future studies on longevity-promoting genes should be carried out in long-lived SSD1-V strains.

scholarly journals A Carboxy-Terminal Monoleucine-Based Motif Participates in the Basolateral Targeting of the Na+/I− Symporter

Endocrinology ◽  
2018 ◽  
Vol 160 (1) ◽  
pp. 156-168 ◽  
Author(s):  
Mariano Martín ◽  
Carlos Pablo Modenutti ◽  
Victoria Peyret ◽  
Romina Celeste Geysels ◽  
Elisabeth Darrouzet ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Thyroid Tissue ◽  
Adaptor Protein ◽  
Site Directed Mutagenesis ◽  
Apical Plasma Membrane ◽  
Thyroid Tumors ◽  
Carboxy Terminus ◽  
Basolateral Plasma Membrane ◽  
Sorting Motif ◽  
Radioiodide Therapy

Abstract The Na+/iodide (I−) symporter (NIS), a glycoprotein expressed at the basolateral plasma membrane of thyroid follicular cells, mediates I− accumulation for thyroid hormonogenesis and radioiodide therapy for differentiated thyroid carcinoma. However, differentiated thyroid tumors often exhibit lower I− transport than normal thyroid tissue (or even undetectable I− transport). Paradoxically, the majority of differentiated thyroid cancers show intracellular NIS expression, suggesting abnormal targeting to the plasma membrane. Therefore, a thorough understanding of the mechanisms that regulate NIS plasma membrane transport would have multiple implications for radioiodide therapy. In this study, we show that the intracellularly facing carboxy-terminus of NIS is required for the transport of the protein to the plasma membrane. Moreover, the carboxy-terminus contains dominant basolateral information. Using internal deletions and site-directed mutagenesis at the carboxy-terminus, we identified a highly conserved monoleucine-based sorting motif that determines NIS basolateral expression. Furthermore, in clathrin adaptor protein (AP)-1B–deficient cells, NIS sorting to the basolateral plasma membrane is compromised, causing the protein to also be expressed at the apical plasma membrane. Computer simulations suggest that the AP-1B subunit σ1 recognizes the monoleucine-based sorting motif in NIS carboxy-terminus. Although the mechanisms by which NIS is intracellularly retained in thyroid cancer remain elusive, our findings may open up avenues for identifying molecular targets that can be used to treat radioiodide-refractory thyroid tumors that express NIS intracellularly.

scholarly journals The NHR1 Domain of Neuralized Binds Delta and Mediates Delta Trafficking and Notch Signaling

2007 ◽  
Vol 18 (1) ◽  
pp. 1-13 ◽  
Author(s):  
Cosimo Commisso ◽  
Gabrielle L. Boulianne
Keyword(s):  
Plasma Membrane ◽  
Notch Signaling ◽  
Ubiquitin Ligase ◽  
Protein Interactions ◽  
Neural Development ◽  
Ring Domain ◽  
Protein Protein Interactions ◽  
Necessary And Sufficient ◽  
Conserved Residue ◽  
Notch Ligands

Notch signaling, which is crucial to metazoan development, requires endocytosis of Notch ligands, such as Delta and Serrate. Neuralized is a plasma membrane-associated ubiquitin ligase that is required for neural development and Delta internalization. Neuralized is comprised of three domains that include a C-terminal RING domain and two neuralized homology repeat (NHR) domains. All three domains are conserved between organisms, suggesting that these regions of Neuralized are functionally important. Although the Neuralized RING domain has been shown to be required for Delta ubiquitination, the function of the NHR domains remains elusive. Here we show that neuralized1, a well-characterized neurogenic allele, exhibits a mutation in a conserved residue of the NHR1 domain that results in mislocalization of Neuralized and defects in Delta binding and internalization. Furthermore, we describe a novel isoform of Neuralized and show that it is recruited to the plasma membrane by Delta and that this is mediated by the NHR1 domain. Finally, we show that the NHR1 domain of Neuralized is both necessary and sufficient to bind Delta. Altogether, our data demonstrate that NHR domains can function in facilitating protein–protein interactions and in the case of Neuralized, mediate binding to its ubiquitination target, Delta.

scholarly journals SLAP2 adaptor binding disrupts c-CBL autoinhibition to activate ubiquitin ligase function

2020 ◽  
Author(s):  
Leanne E. Wybenga-Groot ◽  
Andrea J. Tench ◽  
Craig D. Simpson ◽  
Jonathan St. Germain ◽  
Brian Raught ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Binding Site ◽  
Ubiquitin Ligase ◽  
Adaptor Proteins ◽  
Negative Regulator ◽  
Alternative Mechanism ◽  
Kinase Signaling ◽  
Tyrosine Kinase Signaling ◽  
Ligase Function

AbstractCBL is a RING type E3 ubiquitin ligase that functions as a negative regulator of tyrosine kinase signaling and loss of CBL E3 function is implicated in several forms of leukemia. The Src-like adaptor proteins (SLAP/SLAP2) bind to CBL and are required for CBL-dependent downregulation of antigen receptor, cytokine receptor, and receptor tyrosine kinase signaling. Despite the established role of SLAP/SLAP2 in regulating CBL activity, the nature of the interaction and the mechanisms involved are not known. To understand the molecular basis of the interaction between SLAP/SLAP2 and CBL, we solved the crystal structure of CBL tyrosine kinase binding domain (TKBD) in complex with SLAP2. The carboxy-terminal region of SLAP2 adopts an α-helical structure which binds in a cleft between the 4H, EF-hand, and SH2 domains of the TKBD. This SLAP2 binding site is remote from the canonical TKBD phospho-tyrosine peptide binding site but overlaps with a region important for stabilizing CBL in its autoinhibited conformation. In addition, binding of SLAP2 to CBL in vitro activates the ubiquitin ligase function of autoinhibited CBL. Disruption of the CBL/SLAP2 interface through mutagenesis demonstrated a role for this protein-protein interaction in regulation of CBL E3 ligase activity in cells. Our results reveal that SLAP2 binding to a regulatory cleft of the TKBD provides an alternative mechanism for activation of CBL ubiquitin ligase function.

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