scholarly journals A Large PEST-like Sequence Directs the Ubiquitination, Endocytosis, and Vacuolar Degradation of the Yeast a-Factor Receptor

1998 ◽  
Vol 142 (4) ◽  
pp. 949-961 ◽  
Author(s):  
Amy F. Roth ◽  
Daniel M. Sullivan ◽  
Nicholas G. Davis
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Broad Class ◽  
Surface Protein ◽  
Sequence Motif ◽  
Plasma Membrane Atpase ◽  
Alanine Scanning Mutagenesis ◽  
Entire Interval ◽  
Sequence Elements ◽  
In The Wild

The yeast a-factor receptor (encoded by STE3) is subject to two modes of endocytosis, a ligand-dependent endocytosis as well as a constitutive, ligand-independent mode. Both modes are associated with receptor ubiquitination (Roth, A.F., and N.G. Davis. 1996. J. Cell Biol. 134:661–674) and both depend on sequence elements within the receptor's regulatory, cytoplasmically disposed, COOH-terminal domain (CTD). Here, we concentrate on the Ste3p sequences required for constitutive endocytosis. Constitutive endocytosis is rapid. Receptor is synthesized, delivered to the cell surface, endocytosed, and then delivered to the vacuole where it is degraded, all with a t1/2 of 15 min. Deletion analysis has defined a 36-residue-long sequence mapping near the COOH-terminal end of the Ste3p CTD that is the minimal sequence required for this rapid turnover. Deletions intruding into this interval block or severely slow the rate of endocytic turnover. Moreover, the same 36-residue sequence directs receptor ubiquitination. Mutants deleted for this sequence show undetectable levels of ubiquitination, and mutants having intermediate endocytosis defects show a correlated reduced level of ubiquitination. Not only necessary for ubiquitination and endocytosis, this sequence also is sufficient. When transplanted to a stable cell surface protein, the plasma membrane ATPase Pma1p, the 36-residue STE3 signal directs both ubiquitination of the PMA1-STE3 fusion protein as well as its endocytosis and consequent vacuolar degradation. Alanine scanning mutagenesis across the 36-residue-long interval highlights its overall complexity—no singular sequence motif or signal is found, instead required sequence elements distribute throughout the entire interval. The high proportion of acidic and hydroxylated amino acid residues in this interval suggests a similarity to PEST sequences—a broad class of sequences which have been shown to direct the ubiquitination and subsequent proteosomal degradation of short-lived nuclear and cytoplasmic proteins. A likely possibility, therefore, is that this sequence, responsible for both endocytosis and ubiquitination, may be first and foremost a ubiquitination signal. Finally, we present evidence suggesting that the true signal in the wild-type receptor extends beyond the 36-residue-long sequence defined as a minimal signal to include contiguous PEST-like sequences which extend another 21 residues to the COOH terminus of Ste3p. Together with sequences identified in two other yeast plasma membrane proteins, the STE3 sequence defines a new class of ubiquitination/endocytosis signal.

scholarly journals Biochemical analysis of connexin43 intracellular transport, phosphorylation, and assembly into gap junctional plaques.

1991 ◽  
Vol 115 (5) ◽  
pp. 1357-1374 ◽  
Author(s):  
L S Musil ◽  
D A Goodenough
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Gap Junction ◽  
Intracellular Transport ◽  
Surface Protein ◽  
Immunohistochemical Localization ◽  
Junctional Communication ◽  
Junction Protein ◽  
Gap Junctional ◽  
Triton X

We previously demonstrated that the gap junction protein connexin43 is translated as a 42-kD protein (connexin43-NP) that is efficiently phosphorylated to a 46,000-Mr species (connexin43-P2) in gap junctional communication-competent, but not in communication-deficient, cells. In this study, we used a combination of metabolic radiolabeling and immunoprecipitation to investigate the assembly of connexin43 into gap junctions and the relationship of this event to phosphorylation of connexin43. Examination of the detergent solubility of connexin43 in communication-competent NRK cells revealed that processing of connexin43 to the P2 form was accompanied by acquisition of resistance to solubilization in 1% Triton X-100. Immunohistochemical localization of connexin43 in Triton-extracted NRK cells demonstrated that connexin43-P2 (Triton-insoluble) was concentrated in gap junctional plaques, whereas connexin43-NP (Triton-soluble) was predominantly intracellular. Using either a 20 degrees C intracellular transport block or cell-surface protein biotinylation, we determined that connexin43 was transported to the plasma membrane in the Triton-soluble connexin43-NP form. Cell-surface biotinylated connexin43-NP was processed to Triton-insoluble connexin43-P2 at 37 degrees C. Connexin43-NP was also transported to the plasma membrane in communication defective, gap junction-deficient S180 and L929 cells but was not processed to Triton-insoluble connexin43-P2. Taken together, these results demonstrate that gap junction assembly is regulated after arrival of connexin43 at the plasma membrane and is temporally associated with acquisition of insolubility in Triton X-100 and phosphorylation to the connexin43-P2 form.

scholarly journals Targeting of the yeast plasma membrane [H+]ATPase: a novel gene AST1 prevents mislocalization of mutant ATPase to the vacuole.

1995 ◽  
Vol 128 (1) ◽  
pp. 39-49 ◽  
Author(s):  
A Chang ◽  
G R Fink
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Growth Defect ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Plasma Membrane Atpase ◽  
Novel Gene ◽  
Multicopy Suppressors ◽  
Proteinase A ◽  
Synthetic Growth

We have characterized a class of mutations in PMA1, (encoding plasma membrane ATPase) that is ideal for the analysis of membrane targeting in Saccharomyces cerevisiae. This class of pma1 mutants undergoes growth arrest at the restrictive temperature because newly synthesized ATPase fails to be targeted to the cell surface. Instead, mutant ATPase is delivered to the vacuole, where it is degraded. Delivery to the vacuole occurs without previous arrival at the plasma membrane because degradation of mutant ATPase is not prevented when internalization from the cell surface is blocked. Disruption of PEP4, encoding vacuolar proteinase A, blocks ATPase degradation, but fails to restore growth because the ATPase is still improperly targeted. One of these pma1 mutants was used to select multicopy suppressors that would permit growth at the nonpermissive temperature. A novel gene, AST1, identified by this selection, suppresses several pma1 alleles defective for targeting. The basis for suppression is that multicopy AST1 causes rerouting of mutant ATPase from the vacuole to the cell surface. pma1 mutants deleted for AST1 have a synthetic growth defect at the permissive temperature, providing genetic evidence for interaction between AST1 and PMA1. Ast1 is a cytoplasmic protein that associates with membranes, and is localized to multiple compartments, including the plasma membrane. The identification of AST1 homologues suggests that Ast1 belongs to a novel family of proteins that participates in membrane traffic.

scholarly journals Exomer Is Part of a Hub Where Polarized Secretion and Ionic Stress Connect

2021 ◽  
Vol 12 ◽  
Author(s):  
Sandra Moro ◽  
Esteban Moscoso-Romero ◽  
Abhishek Poddar ◽  
Jose M. Mulet ◽  
Pilar Perez ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Transient Receptor Potential ◽  
Ion Homeostasis ◽  
Wild Type ◽  
Plasma Membrane Atpase ◽  
General Role ◽  
Membrane Hyperpolarization ◽  
Polarized Secretion ◽  
In The Wild ◽  
Golgi Network

Plasma membrane and membranous organelles contribute to the physiology of the Eukaryotic cell by participating in vesicle trafficking and the maintenance of ion homeostasis. Exomer is a protein complex that facilitates vesicle transport from the trans-Golgi network to the plasma membrane, and its absence leads to the retention of a set of selected cargoes in this organelle. However, this retention does not explain all phenotypes observed in exomer mutants. The Schizosaccharomyces pombe exomer is composed of Cfr1 and Bch1, and cfr1Δ and bch1Δ were sensitive to high concentrations of potassium salts but not sorbitol, which showed sensitivity to ionic but not osmotic stress. Additionally, the activity of the plasma membrane ATPase was higher in exomer mutants than in the wild-type, pointing to membrane hyperpolarization, which caused an increase in intracellular K+ content and mild sensitivity to Na+, Ca2+, and the aminoglycoside antibiotic hygromycin B. Moreover, in response to K+ shock, the intracellular Ca2+ level of cfr1Δ cells increased significantly more than in the wild-type, likely due to the larger Ca2+ spikes in the mutant. Microscopy analyses showed a defective endosomal morphology in the mutants. This was accompanied by an increase in the intracellular pools of the K+ exporting P-type ATPase Cta3 and the plasma membrane Transient Receptor Potential (TRP)-like Ca2+ channel Pkd2, which were partially diverted from the trans-Golgi network to the prevacuolar endosome. Despite this, most Cta3 and Pkd2 were delivered to the plasma membrane at the cell growing sites, showing that their transport from the trans-Golgi network to the cell surface occurred in the absence of exomer. Nevertheless, shortly after gene expression in the presence of KCl, the polarized distribution of Cta3 and Pkd2 in the plasma membrane was disturbed in the mutants. Finally, the use of fluorescent probes suggested that the distribution and dynamics of association of some lipids to the plasma membrane in the presence of KCl were altered in the mutants. Thus, exomer participation in the response to K+ stress was multifaceted. These results supported the notion that exomer plays a general role in protein sorting at the trans-Golgi network and in polarized secretion, which is not always related to a function as a selective cargo adaptor.

scholarly journals Extracellular pH Dynamically Controls Cell Surface Delivery of Functional TRPV5 Channels

2006 ◽  
Vol 27 (4) ◽  
pp. 1486-1494 ◽  
Author(s):  
Tim T. Lambers ◽  
Elena Oancea ◽  
Theun de Groot ◽  
Catalin N. Topala ◽  
Joost G. Hoenderop ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Transient Receptor Potential ◽  
Trp Channels ◽  
Surface Protein ◽  
Receptor Potential ◽  
Transport Processes ◽  
Extracellular Ph ◽  
Cell Surface Expression ◽  
Transient Receptor Potential Vanilloid

ABSTRACT Extracellular pH has long been known to affect the rate and magnitude of ion transport processes among others via regulation of ion channel activity. The Ca2+-selective transient receptor potential vanilloid 5 (TRPV5) channel constitutes the apical entry gate in Ca2+-transporting cells, contributing significantly to the overall Ca2+ balance. Here, we demonstrate that extracellular pH determines the cell surface expression of TRPV5 via a unique mechanism. By a comprehensive approach using total internal reflection fluorescence microscopy, cell surface protein labeling, electrophysiology, 45Ca2+ uptake assays, and functional channel recovery after chemobleaching, this study shows that upon extracellular alkalinization, a pool of TRPV5-containing vesicles is rapidly recruited to the cell surface without collapsing into the plasma membrane. These vesicles contain functional TRPV5 channels since extracellular alkalinization is accompanied by increased TRPV5 activity. Conversely, upon subsequent extracellular acidification, vesicles are retrieved from the plasma membrane, simultaneously resulting in decreased TRPV5 activity. Thus, TRPV5 accesses the extracellular compartment via transient openings of vesicles, suggesting that rapid responses of constitutive active TRP channels to physiological stimuli rely on vesicular “kiss and linger” interactions with the plasma membrane.

scholarly journals Sialidase NEU3 is a peripheral membrane protein localized on the cell surface and in endosomal structures

2007 ◽  
Vol 408 (2) ◽  
pp. 211-219 ◽  
Author(s):  
Gabriele Zanchetti ◽  
Paolo Colombi ◽  
Marta Manzoni ◽  
Luigi Anastasia ◽  
Luigi Caimi ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Cell Surface ◽  
Surface Protein ◽  
Experimental Conditions ◽  
Cellular Processes ◽  
Endosomal Compartment ◽  
Outer Leaflet ◽  
Peripheral Membrane Protein ◽  
The Moment

Sialidase NEU3 is also known as the plasma-membrane-associated form of mammalian sialidases, exhibiting a high substrate specificity towards gangliosides. In this respect, sialidase NEU3 modulates cell-surface biological events and plays a pivotal role in different cellular processes, including cell adhesion, recognition and differentiation. At the moment, no detailed studies concerning the subcellular localization of NEU3 are available, and the mechanism of its association with cellular membranes is still unknown. In the present study, we have demonstrated that sialidase NEU3, besides its localization at the plasma membrane, is present in intracellular structures at least partially represented by a subset of the endosomal compartment. Moreover, we have shown that NEU3 present at the plasma membrane is internalized and locates then to the recycling endosomal compartment. The enzyme is associated with the outer leaflet of the plasma membrane, as shown by selective cell-surface protein biotinylation. This evidence is in agreement with the ability of NEU3 to degrade gangliosides inserted into the plasma membrane of adjacent cells. Moreover, the mechanism of the protein association with the lipid bilayer was elucidated by carbonate extraction. Under these experimental conditions, we have succeeded in solubilizing NEU3, thus demonstrating that the enzyme is a peripheral membrane protein. In addition, Triton X-114 phase separation demonstrates further the hydrophilic nature of the protein. Overall, these results provide important information about the biology of NEU3, the most studied member of the mammalian sialidase family.

scholarly journals An Endosome-to-Plasma Membrane Pathway Involved in Trafficking of a Mutant Plasma Membrane ATPase in Yeast

2000 ◽  
Vol 11 (2) ◽  
pp. 579-592 ◽  
Author(s):  
Wen-jie Luo ◽  
Amy Chang
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Secretory Pathway ◽  
Protein Sorting ◽  
Temperature Sensitive ◽  
Plasma Membrane Atpase ◽  
Vacuolar Protein Sorting ◽  
Membrane Atpase ◽  
Vacuolar Protein ◽  
Endosomal System

The plasma membrane ATPase, encoded by PMA1, is delivered to the cell surface via the secretory pathway. Previously, we characterized a temperature-sensitive pma1 mutant in which newly synthesized Pma1-7 is not delivered to the plasma membrane but is mislocalized instead to the vacuole at 37°C. Severalvps mutants, which are defective in vacuolar protein sorting, suppress targeting-defective pma1 by allowing mutant Pma1 to move once again to the plasma membrane. In this study, we have analyzed trafficking in the endosomal system by monitoring the movement of Pma1-7 in vps36, vps1, andvps8 mutants. Upon induction of expression, mutant Pma1 accumulates in the prevacuolar compartment in vps36cells. After chase, a fraction of newly synthesized Pma1-7 is delivered to the plasma membrane. In both vps1 andvps8 cells, newly synthesized mutant Pma1 appears in small punctate structures before arrival at the cell surface. Nevertheless, biosynthetic membrane traffic appears to follow different routes in vps8 and vps1: the vacuolar protein-sorting receptor Vps10p is stable in vps8 but not in vps1. Furthermore, a defect in endocytic delivery to the vacuole was revealed in vps8 (andvps36) but not vps1 by endocytosis of the bulk membrane marker FM 4-64. Moreover, in vps8 cells, there is defective down-regulation from the cell surface of the mating receptor Ste3, consistent with persistent receptor recycling from an endosomal compartment to the plasma membrane. These data support a model in which mutant Pma1 is diverted from the Golgi to the surface invps1 cells. We hypothesize that in vps8and vps36, in contrast to vps1, mutant Pma1 moves to the surface via endosomal intermediates, implicating an endosome-to-surface traffic pathway.

scholarly journals Ubiquitin-mediated Targeting of a Mutant Plasma Membrane ATPase, Pma1-7, to the Endosomal/Vacuolar System in Yeast

2004 ◽  
Vol 15 (5) ◽  
pp. 2401-2409 ◽  
Author(s):  
Maddalena Pizzirusso ◽  
Amy Chang
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Ubiquitin Ligase ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Plasma Membrane Atpase ◽  
Membrane Atpase ◽  
Endosomal Pathway ◽  
Quality Control Mechanism ◽  
Vacuolar System

Pma1-7 is a mutant plasma membrane ATPase that is impaired in targeting to the cell surface at 37°C and is delivered instead to the endosomal/vacuolar pathway for degradation. We have proposed that Pma1-7 is a substrate for a Golgibased quality control mechanism. By contrast with wild-type Pma1, Pma1-7 is ubiquitinated. Ubiquitination and endosomal targeting of Pma1-7 is dependent on the Rsp5-Bul1-Bul2 ubiquitin ligase protein complex but not the transmembrane ubiquitin ligase Tul1. Analysis of Pma1-7 ubiquitination in mutants blocked in protein transport at various steps of the secretory pathway suggests that ubiquitination occurs after ER exit but before endosomal entry. In the absence of ubiquitination in rsp5-1 cells, Pma1-7 is delivered to the cell surface and remains stable. Nevertheless, Pma1-7 remains impaired in association with detergent-insoluble glycolipid-enriched complexes in rsp5-1 cells, suggesting that ubiquitination is not the cause of Pma1-7 exclusion from rafts. In vps1 cells in which protein transport into the endosomal pathway is blocked, Pma1-7 is routed to the cell surface. On arrival at the plasma membrane in vps1 cells, Pma1-7 remains stable and its ubiquitination disappears, suggesting deubiquitination activity at the cell surface. We suggest that Pma1-7 sorting and fate are regulated by ubiquitination.

scholarly journals The Chymotrypsin-Like Protease Complex of Treponema denticola ATCC 35405 Mediates Fibrinogen Adherence and Degradation

2007 ◽  
Vol 75 (9) ◽  
pp. 4364-4372 ◽  
Author(s):  
Caroline V. Bamford ◽  
J. Christopher Fenno ◽  
Howard F. Jenkinson ◽  
David Dymock
Keyword(s):  
Cell Surface ◽  
Mutant Strain ◽  
Surface Protein ◽  
Fluid Phase ◽  
Treponema Denticola ◽  
Wild Type ◽  
Chromatography Analysis ◽  
Fibrinogen Binding ◽  
In The Wild ◽  
Major Surface

ABSTRACT Treponema denticola is an anaerobic spirochete strongly associated with human periodontal disease. T. denticola bacteria interact with a range of host tissue proteins, including fibronectin, laminin, and fibrinogen. The latter localizes in the extracellular matrix where tissue damage has occurred, and interactions with fibrinogen may play a key role in T. denticola colonization of the damaged sites. T. denticola ATCC 35405 showed saturable binding of fluid-phase fibrinogen to the cell surface and saturable adherence to immobilized fibrinogen. Levels of fibrinogen binding were enhanced in the presence of the serine protease inhibitor phenylmethylsulfonyl fluoride. The Aα and Bβ chains of fibrinogen, but not the γ chains, were specifically recognized by T. denticola. Following fibrinogen affinity chromatography analysis of cell surface extracts, a major fibrinogen-binding component (polypeptide molecular mass, ∼100 kDa), which also degraded fibrinogen, was purified. Upon heating at 100°C, the polypeptide was dissociated into three components (apparent molecular masses, 80, 48, and 45 kDa) that did not individually bind or degrade fibrinogen. The native 100-kDa polypeptide complex was identified as chymotrypsin-like protease (CTLP), or dentilisin. In an isogenic CTLP− mutant strain, CKE, chymotrypsin-like activity was reduced >90% compared to that in the wild type and fibrinogen binding and hydrolysis were ablated. Isogenic mutant strain MHE, deficient in the production of Msp (major surface protein), showed levels of CTLP reduced 40% relative to those in the wild type and exhibited correspondingly reduced levels of fibrinogen binding and proteolysis. Thrombin clotting times in the presence of wild-type T. denticola cells, but not strain CKE (CTLP−) cells, were extended. These results suggest that interactions of T. denticola with fibrinogen, which may promote colonization and modulate hemostasis, are mediated principally by CTLP.

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