The C2 domain of the ubiquitin ligase Rsp5 is required for ubiquitination of the endocytic protein Rvs167 upon change of nitrogen source

ABSTRACT Ubiquitination is a key signal for endocytosis of proteins on the plasma membrane. The ubiquitin ligase Rsp5 of Saccharomyces cerevisiae, which contains an amino-terminal membrane-binding C2 domain, three substrate-recognizing tryptophan-tryptophan (WW) domains and a carboxyl-terminal catalytic homologous to the E6-AP carboxyl terminus (HECT) domain, can ubiquitinate plasma membrane proteins directing them for endocytosis. Here, we examined the roles of the C2 domain in endocytosis for the downregulation of the general amino acid permease Gap1, which is one of nitrogen-regulated permeases in S. cerevisiae. First, we constructed several rsp5 mutants producing Rsp5 variants without the C2 domain or with amino acid changes of membrane-binding lysine residues. These mutants showed defects in endocytosis of Gap1 in response to a preferred nitrogen source. Intriguingly, we found that ubiquitination of Gap1 in these mutant cells was highly similar to that in wild-type cells during endocytosis. These results indicate that the C2 domain is essential for endocytosis but not for ubiquitination of substrates such as Gap1. Moreover, genetic and biochemical analyses showed that the endocytic protein Rvs167 was ubiquitinated via Rsp5 and the C2 domain was required for efficient ubiquitination in response to a preferred nitrogen source. Here, we propose a mechanism for the C2 domain-mediated endocytosis of plasma membrane permeases.

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Components of a Ubiquitin Ligase Complex Specify Polyubiquitination and Intracellular Trafficking of the General Amino Acid Permease

The Journal of Cell Biology ◽

10.1083/jcb.153.4.649 ◽

2001 ◽

Vol 153 (4) ◽

pp. 649-662 ◽

Cited By ~ 206

Author(s):

Stephen B. Helliwell ◽

Sascha Losko ◽

Chris A. Kaiser

Keyword(s):

Plasma Membrane ◽

Amino Acid ◽

Nitrogen Source ◽

Ubiquitin Ligase ◽

Amino Acid Uptake ◽

Acid Uptake ◽

Amino Acid Permease ◽

Intracellular Targeting ◽

Ubiquitin Ligase Complex ◽

General Amino Acid Permease

Gap1p, the general amino acid permease of Saccharomyces cerevisiae, is regulated by intracellular sorting decisions that occur in either Golgi or endosomal compartments. Depending on nitrogen source, Gap1p is transported to the plasma membrane, where it functions for amino acid uptake, or to the vacuole, where it is degraded. We found that overexpression of Bul1p or Bul2p, two nonessential components of the Rsp5p E3–ubiquitin ligase complex, causes Gap1p to be sorted to the vacuole regardless of nitrogen source. The double mutant bul1Δ bul2Δ has the inverse phenotype, causing Gap1p to be delivered to the plasma membrane more efficiently than in wild-type cells. In addition, bul1Δ bul2Δ can reverse the effect of lst4Δ, a mutation that normally prevents Gap1p from reaching the plasma membrane. Evaluation of Gap1p ubiquitination revealed a prominent polyubiquitinated species that was greatly diminished in a bul1Δ bul2Δ mutant. Both a rsp5-1 mutant and a COOH-terminal truncation of Gap1p behave as bul1Δ bul2Δ, causing constitutive delivery of Gap1p to the plasma membrane and decreasing Gap1p polyubiquitination. These results indicate that Bul1p and Bul2p, together with Rsp5p, generate a polyubiquitin signal on Gap1p that specifies its intracellular targeting to the vacuole.

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

Bioscience Biotechnology and Biochemistry ◽

10.1093/bbb/zbab028 ◽

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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Specific Translocation of Protein Kinase Cα to the Plasma Membrane Requires Both Ca2+ and PIP2 Recognition by Its C2 Domain

Molecular Biology of the Cell ◽

10.1091/mbc.e05-06-0499 ◽

2006 ◽

Vol 17 (1) ◽

pp. 56-66 ◽

Cited By ~ 77

Author(s):

John H. Evans ◽

Diana Murray ◽

Christina C. Leslie ◽

Joseph J. Falke

Keyword(s):

Plasma Membrane ◽

Protein Kinase ◽

Membrane Binding ◽

Membrane Lipid ◽

C2 Domain ◽

Plasma Membrane Lipid ◽

Protein Kinase Cα ◽

Golgi Network

The C2 domain of protein kinase Cα (PKCα) controls the translocation of this kinase from the cytoplasm to the plasma membrane during cytoplasmic Ca2+ signals. The present study uses intracellular coimaging of fluorescent fusion proteins and an in vitro FRET membrane-binding assay to further investigate the nature of this translocation. We find that Ca2+-activated PKCα and its isolated C2 domain localize exclusively to the plasma membrane in vivo and that a plasma membrane lipid, phosphatidylinositol-4,5-bisphosphate (PIP2), dramatically enhances the Ca2+-triggered binding of the C2 domain to membranes in vitro. Similarly, a hybrid construct substituting the PKCα Ca2+-binding loops (CBLs) and PIP2 binding site (β-strands 3–4) into a different C2 domain exhibits native Ca2+-triggered targeting to plasma membrane and recognizes PIP2. Conversely, a hybrid containing the CBLs but lacking the PIP2 site translocates primarily to trans-Golgi network (TGN) and fails to recognize PIP2. Similarly, PKCα C2 domains possessing mutations in the PIP2 site target primarily to TGN and fail to recognize PIP2. Overall, these findings demonstrate that the CBLs are essential for Ca2+-triggered membrane binding but are not sufficient for specific plasma membrane targeting. Instead, targeting specificity is provided by basic residues on β-strands 3–4, which bind to plasma membrane PIP2.

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Casein Kinase I Regulates Membrane Binding by ARF GAP1

Molecular Biology of the Cell ◽

10.1091/mbc.e02-04-0189 ◽

2002 ◽

Vol 13 (8) ◽

pp. 2559-2570 ◽

Cited By ~ 33

Author(s):

Sidney Yu ◽

Michael G. Roth

Keyword(s):

Amino Acid ◽

Secretory Pathway ◽

Protein Transport ◽

Membrane Binding ◽

Casein Kinase ◽

Protein Domain ◽

Amino Terminal ◽

Early Secretory Pathway

ARF GAP1, a 415-amino acid GTPase activating protein (GAP) for ADP-ribosylation factor (ARF) contains an amino-terminal 115-amino acid catalytic domain and no other recognizable features. Amino acids 203–334 of ARF GAP1 were sufficient to target a GFP-fusion protein to Golgi membranes in vivo. When overexpressed in COS-1 cells, this protein domain inhibited protein transport between the ER and Golgi and, in vitro, competed with the full-length ARF GAP1 for binding to membranes. Membrane binding by ARF GAP1 in vitro was increased by a factor in cytosol and this increase was inhibited by IC261, an inhibitor selective for casein kinase Iδ (CKIδ), or when cytosol was treated with antibody to CKIδ. The noncatalytic domain of ARF GAP1 was phosphorylated both in vivo and in vitro by CKI. IC261 blocked membrane binding by ARF GAP1 in vivo and inhibited protein transport in the early secretory pathway. Overexpression of a catalytically inactive CKIδ also inhibited the binding of ARF GAP1 to membranes and interfered with protein transport. Thus, a CKI isoform is required for protein traffic through the early secretory pathway and can modulate the amount of ARF GAP1 that can bind to membranes.

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

Clinical Science ◽

10.1042/cs20150554 ◽

2016 ◽

Vol 130 (7) ◽

pp. 499-512 ◽

Cited By ~ 34

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.

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Amino acid permeases require COPII components and the ER resident membrane protein Shr3p for packaging into transport vesicles in vitro.

The Journal of Cell Biology ◽

10.1083/jcb.135.3.585 ◽

1996 ◽

Vol 135 (3) ◽

pp. 585-595 ◽

Cited By ~ 90

Author(s):

M J Kuehn ◽

R Schekman ◽

P O Ljungdahl

Keyword(s):

Plasma Membrane ◽

Amino Acid ◽

Membrane Protein ◽

Integral Membrane Protein ◽

Plasma Membrane Atpase ◽

Amino Acid Permease ◽

Transport Vesicles ◽

Amino Acid Permeases ◽

Histidine Permease

In S. cerevisiae lacking SHR3, amino acid permeases specifically accumulate in membranes of the endoplasmic reticulum (ER) and fail to be transported to the plasma membrane. We examined the requirements of transport of the permeases from the ER to the Golgi in vitro. Addition of soluble COPII components (Sec23/24p, Sec13/31p, and Sar1p) to yeast membrane preparations generated vesicles containing the general amino acid permease. Gap1p, and the histidine permease, Hip1p. Shr3p was required for the packaging of Gap1p and Hip1p but was not itself incorporated into transport vesicles. In contrast, the packaging of the plasma membrane ATPase, Pma1p, and the soluble yeast pheromone precursor, glycosylated pro alpha factor, was independent of Shr3p. In addition, we show that integral membrane and soluble cargo colocalize in transport vesicles, indicating that different types of cargo are not segregated at an early step in secretion. Our data suggest that specific ancillary proteins in the ER membrane recruit subsets of integral membrane protein cargo into COPII transport vesicles.

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Genetic and Biochemical Analysis of the Yeast Plasma Membrane Ssy1p-Ptr3p-Ssy5p Sensor of Extracellular Amino Acids

Molecular and Cellular Biology ◽

10.1128/mcb.21.3.814-826.2001 ◽

2001 ◽

Vol 21 (3) ◽

pp. 814-826 ◽

Cited By ~ 114

Author(s):

Hanna Forsberg ◽

Per O. Ljungdahl

Keyword(s):

Amino Acids ◽

Plasma Membrane ◽

Amino Acid ◽

Membrane System ◽

Dominant Negative ◽

Amino Acid Permease ◽

Yeast Plasma Membrane ◽

Sensing System ◽

Amino Acid Availability ◽

Cell Extracts

ABSTRACT Ssy1p and Ptr3p are known components of a yeast plasma membrane system that functions to sense the presence of amino acids in the extracellular environment. In response to amino acids, this sensing system initiates metabolic signals that ultimately regulate the functional expression of several amino acid-metabolizing enzymes and transport proteins, including multiple, genetically distinct amino acid permeases. We have found that SSY5 encodes a third component of this amino acid sensing system. Mutations inSSY5 manifest phenotypes that are indistinguishable from those resulting from either single ssy1 andptr3 mutations or ssy5 ssy1 and ssy5 ptr3 double mutations. Although Ssy5p is predicted to be a soluble protein, it exhibits properties indicating that it is a peripherally associated plasma membrane protein. Each of the three sensor components, Ssy1p, Ptr3p, and Ssy5p, adopts conformations and modifications that are dependent upon the availability of amino acids and on the presence of the other two components. These results suggest that these components function as part of a sensor complex localized to the plasma membrane. Consistent with a sensor complex, the overexpression of SSY1 or the unique N-terminal extension of this amino acid permease homologue inactivates the amino acid sensor in a dominant-negative manner. Each of the components of the Ssy1p-Ptr3p-Ssy5p (SPS) signaling system undergoes rapid physical changes, reflected in altered electrophoretic mobility, when leucine is added to cells grown in media lacking amino acids. Furthermore, the levels of each SPS sensor component present in whole-cell extracts diminish upon leucine addition. The rapid physical alterations and reduced levels of sensor components are consistent with their being downregulated in response to amino acid availability. These results reveal the dynamic nature of the amino acid-initiated signals transduced by the SPS sensor.

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Different Ubiquitin Signals Act at the Golgi and Plasma Membrane to Direct GAP1 Trafficking

Molecular Biology of the Cell ◽

10.1091/mbc.e07-06-0627 ◽

2008 ◽

Vol 19 (7) ◽

pp. 2962-2972 ◽

Cited By ~ 39

Author(s):

April L. Risinger ◽

Chris A. Kaiser

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acids ◽

Plasma Membrane ◽

Amino Acid ◽

Protein Sorting ◽

High Capacity ◽

Amino Acid Permease ◽

Acid Addition ◽

Amino Acid Levels ◽

General Amino Acid Permease

The high capacity general amino acid permease, Gap1p, in Saccharomyces cerevisiae is distributed between the plasma membrane and internal compartments according to availability of amino acids. When internal amino acid levels are low, Gap1p is localized to the plasma membrane where it imports available amino acids from the medium. When sufficient amino acids are imported, Gap1p at the plasma membrane is endocytosed and newly synthesized Gap1p is delivered to the vacuole; both sorting steps require Gap1p ubiquitination. Although it has been suggested that identical trans-acting factors and Gap1p ubiquitin acceptor sites are involved in both processes, we define unique requirements for each of the ubiquitin-mediated sorting steps involved in delivery of Gap1p to the vacuole upon amino acid addition. Our finding that distinct ubiquitin-mediated sorting steps employ unique trans-acting factors, ubiquitination sites on Gap1p, and types of ubiquitination demonstrates a previously unrecognized level of specificity in ubiquitin-mediated protein sorting.

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