scholarly journals Clathrin adaptors mediate two sequential pathways of intra-Golgi recycling

2021 ◽  
Vol 221 (1) ◽  
Author(s):  
Jason C. Casler ◽  
Natalie Johnson ◽  
Adam H. Krahn ◽  
Areti Pantazopoulou ◽  
Kasey J. Day ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Golgi Apparatus ◽  
Transmembrane Proteins ◽  
Membrane Traffic ◽  
Yeast Saccharomyces Cerevisiae ◽  
Clathrin Adaptor ◽  
Pass Through ◽  
Kinetics Of

The pathways of membrane traffic within the Golgi apparatus are not fully known. This question was addressed using the yeast Saccharomyces cerevisiae, in which the maturation of individual Golgi cisternae can be visualized. We recently proposed that the AP-1 clathrin adaptor mediates intra-Golgi recycling late in the process of cisternal maturation. Here, we demonstrate that AP-1 cooperates with the Ent5 clathrin adaptor to recycle a set of Golgi transmembrane proteins, including some that were previously thought to pass through endosomes. This recycling can be detected by removing AP-1 and Ent5, thereby diverting the AP-1/Ent5–dependent Golgi proteins into an alternative recycling loop that involves traffic to the plasma membrane followed by endocytosis. Unexpectedly, various AP-1/Ent5–dependent Golgi proteins show either intermediate or late kinetics of residence in maturing cisternae. We infer that the AP-1/Ent5 pair mediates two sequential intra-Golgi recycling pathways that define two classes of Golgi proteins. This insight can explain the polarized distribution of transmembrane proteins in the Golgi.

scholarly journals Clathrin adaptors mediate two sequential pathways of intra-Golgi recycling

2021 ◽  
Author(s):  
Jason C. Casler ◽  
Adam H. Krahn ◽  
Areti Pantazopoulou ◽  
Natalie Johnson ◽  
Kasey J. Day ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Golgi Apparatus ◽  
Transmembrane Proteins ◽  
Membrane Traffic ◽  
Clathrin Adaptor ◽  
Kinetics Of

The pathways of membrane traffic within the Golgi apparatus are not fully known. This question was addressed using the yeast Saccharomyces cerevisiae, in which the maturation of individual Golgi cisternae can be visualized. We recently proposed that the AP-1 clathrin adaptor mediates intra-Golgi recycling late in the process of cisternal maturation. Here, we demonstrate that AP-1 cooperates with the Ent5 clathrin adaptor to recycle a set of Golgi transmembrane proteins. This recycling can be detected by removing AP-1 and Ent5, thereby diverting the AP-1/Ent5-dependent Golgi proteins into an alternative recycling loop that involves traffic to the plasma membrane followed by endocytosis. Unexpectedly, various AP-1/Ent5-dependent Golgi proteins show either intermediate or late kinetics of residence in maturing cisternae. We infer that the AP-1/Ent5 pair mediates two sequential intra-Golgi recycling pathways that define two classes of Golgi proteins. This insight can explain the polarized distribution of transmembrane proteins in the Golgi.

scholarly journals The a-factor transporter (STE6 gene product) and cell polarity in the yeast Saccharomyces cerevisiae.

1993 ◽  
Vol 120 (5) ◽  
pp. 1203-1215 ◽  
Author(s):  
K Kuchler ◽  
H G Dohlman ◽  
J Thorner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Gene Product ◽  
Cell Polarity ◽  
Polyclonal Antibodies ◽  
Apparent Molecular Weight ◽  
Subcellular Fractionation ◽  
Mating Pheromone ◽  
Yeast Saccharomyces Cerevisiae ◽  
Factor Secretion

STE6 gene product is required for secretion of the lipopeptide mating pheromone a-factor by Saccharomyces cerevisiae MATa cells. Radiolabeling and immunoprecipitation, either with specific polyclonal antibodies raised against a TrpE-Ste6 fusion protein or with mAbs that recognize c-myc epitopes in fully functional epitope-tagged Ste6 derivatives, demonstrated that Ste6 is a 145-kD phosphoprotein. Subcellular fractionation, various extraction procedures, and immunoblotting showed that Ste6 is an intrinsic plasma membrane-associated protein. The apparent molecular weight of Ste6 was unaffected by tunicamycin treatment, and the radiolabeled protein did not bind to concanavalin A, indicating that Ste6 is not glycosylated and that glycosylation is not required either for its membrane delivery or its function. The amino acid sequence of Ste6 predicts two ATP-binding folds; correspondingly, Ste6 was photoaffinity-labeled specifically with 8-azido-[alpha-32P]ATP. Indirect immunofluorescence revealed that in exponentially growing MATa cells, the majority of Ste6 showed a patchy distribution within the plasma membrane, but a significant fraction was found concentrated in a number of vesicle-like bodies subtending the plasma membrane. In contrast, in MATa cells exposed to the mating pheromone alpha-factor, which markedly induced Ste6 production, the majority of Ste6 was incorporated into the plasma membrane within the growing tip of the elongating cells. The highly localized insertion of this transporter may establish pronounced anisotropy in a-factor secretion from the MATa cell, and thereby may contribute to the establishment of the cell polarity which restricts partner selection and cell fusion during mating to one MAT alpha cell.

scholarly journals Evaluation of the Fermentation Dynamics of Commercial Baker’s Yeast in Presence of Pistachio Powder to Produce Lysine-Enriched Breads

Fermentation ◽  
2019 ◽  
Vol 6 (1) ◽  
pp. 2 ◽  
Author(s):  
Antonio Alfonzo ◽  
Raimondo Gaglio ◽  
Marcella Barbera ◽  
Nicola Francesca ◽  
Giancarlo Moschetti ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Density ◽  
Titratable Acidity ◽  
Baker’S Yeast ◽  
Baker's Yeast ◽  
Yeast Saccharomyces Cerevisiae ◽  
Promising Strategy ◽  
Total Titratable Acidity ◽  
Bread Production ◽  
Kinetics Of

The present work was carried out to evaluate the microbiological, physicochemical, and sensory characteristics of fortified pistachio breads. Pistachio powder (5% w/w) was added to flour or semolina and fermented by a commercial baker’s yeast (Saccharomyces cerevisiae). Pistachio powder did not influence the biological leavening of the doughs. The kinetics of pH and total titratable acidity (TTA) during dough fermentation showed that the leavening process occurred similarly for all trials. The concentration of yeasts increased during fermentation and reached levels of 108 CFU/g after 2 h. Pistachio powder decreased the height and softness of the final breads and increased cell density of the central slices. The amount of lysine after baking increased in pistachio breads and this effect was stronger for semolina rather than flour trials. Sensory evaluation indicated that fortified breads processed from semolina were those more appreciated by the judges. This work clearly indicated that the addition of pistachio powder in bread production represents a promising strategy to increase the availability of lysine in cereal-based fermented products.

scholarly journals Cell surface recycling in yeast: mechanisms and machineries

2016 ◽  
Vol 44 (2) ◽  
pp. 474-478 ◽  
Author(s):  
Chris MacDonald ◽  
Robert C. Piper
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Membrane Protein ◽  
Cell Surface ◽  
Mammalian Cells ◽  
Genetic System ◽  
Integral Membrane Protein ◽  
Protein Activity ◽  
Yeast Saccharomyces Cerevisiae ◽  
Central Process

Sorting internalized proteins and lipids back to the cell surface controls the supply of molecules throughout the cell and regulates integral membrane protein activity at the surface. One central process in mammalian cells is the transit of cargo from endosomes back to the plasma membrane (PM) directly, along a route that bypasses retrograde movement to the Golgi. Despite recognition of this pathway for decades we are only beginning to understand the machinery controlling this overall process. The budding yeast Saccharomyces cerevisiae, a stalwart genetic system, has been routinely used to identify fundamental proteins and their modes of action in conserved trafficking pathways. However, the study of cell surface recycling from endosomes in yeast is hampered by difficulties that obscure visualization of the pathway. Here we briefly discuss how recycling is likely a more prevalent process in yeast than is widely appreciated and how tools might be built to better study the pathway.

scholarly journals ATP-sensitive K+ channels in a plasma membrane H+-ATPase mutant of the yeast Saccharomyces cerevisiae.

1989 ◽  
Vol 86 (20) ◽  
pp. 7866-7870 ◽  
Author(s):  
J. A. Ramirez ◽  
V. Vacata ◽  
J. H. McCusker ◽  
J. E. Haber ◽  
R. K. Mortimer ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Yeast Saccharomyces Cerevisiae ◽  
K Channels

Amiodarone induces stress responses and calcium flux mediated by the cell wall in Saccharomyces cerevisiae

2009 ◽  
Vol 55 (3) ◽  
pp. 288-303 ◽  
Author(s):  
William E. Courchesne ◽  
Meral Tunc ◽  
Sha Liao
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Wall ◽  
Stress Responses ◽  
Inhibitory Effect ◽  
Calcium Flux ◽  
Yeast Saccharomyces Cerevisiae ◽  
Proteomic Approach ◽  
Amiodarone Treatment ◽  
Kinetics Of

We used a proteomic approach to study effects of amiodarone on cells of the yeast Saccharomyces cerevisiae. Amiodarone has been shown to have antifungal activity in vitro and causes a massive increase in cytoplasmic calcium levels ([Ca2+]cyt). Proteomic analysis of cells exposed to amiodarone show that this drug elicits stress responses and points to involvement of proteins associated with the cell wall. We tested several of those proteins for involvement in the Ca2+ flux. In particular, the amiodarone-induced Ca2+ flux was decreased in bgl2Δ cells, which have altered levels of β-glucan and chitin. The involvement of the cell wall in the Ca2+ flux induced by amiodarone treatment was tested by addition of yeast cell-wall components. While mannan inhibited the rise in [Ca2+]cyt, β-glucan potentiated the Ca2+ flux by 4.5-fold, providing evidence that the cell wall is directly involved in controlling this Ca2+ flux. This conclusion is corroborated by the inhibition of the Ca2+ flux by calcofluor, which is known to bind to cell-wall chitin and inhibit cell growth. Zymolyase treatment altered the kinetics of amiodarone-induced calcium flux and uncoupled the inhibitory effect of calcofluor. These effects demonstrate that the cell-wall β-glucan regulates calcium flux elicited by amiodarone.

scholarly journals A structural mechanism for phosphorylation-dependent inactivation of the AP2 complex

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Edward A Partlow ◽  
Richard W Baker ◽  
Gwendolyn M Beacham ◽  
Joshua S Chappie ◽  
Andres E Leschziner ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Transmembrane Proteins ◽  
Active Conformation ◽  
Genetic Screens ◽  
Structural Mechanism ◽  
C Elegans ◽  
Dependent Inactivation ◽  
Clathrin Adaptor

Endocytosis of transmembrane proteins is orchestrated by the AP2 clathrin adaptor complex. AP2 dwells in a closed, inactive state in the cytosol, but adopts an open, active conformation on the plasma membrane. Membrane-activated complexes are also phosphorylated, but the significance of this mark is debated. We recently proposed that NECAP negatively regulates AP2 by binding open and phosphorylated complexes (Beacham et al., 2018). Here, we report high-resolution cryo-EM structures of NECAP bound to phosphorylated AP2. The site of AP2 phosphorylation is directly coordinated by residues of the NECAP PHear domain that are predicted from genetic screens in C. elegans. Using membrane mimetics to generate conformationally open AP2, we find that a second domain of NECAP binds these complexes and cryo-EM reveals both domains of NECAP engaging closed, inactive AP2. Assays in vitro and in vivo confirm these domains cooperate to inactivate AP2. We propose that phosphorylation marks adaptors for inactivation.

scholarly journals A structural mechanism for phosphorylation-dependent inactivation of the AP2 complex

2019 ◽  
Author(s):  
Edward A. Partlow ◽  
Richard W. Baker ◽  
Gwendolyn M. Beacham ◽  
Joshua S. Chappie ◽  
Andres E. Leschziner ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Transmembrane Proteins ◽  
Active Conformation ◽  
Genetic Screens ◽  
Structural Mechanism ◽  
C Elegans ◽  
Dependent Inactivation ◽  
Clathrin Adaptor

AbstractEndocytosis of transmembrane proteins is orchestrated by the AP2 clathrin adaptor complex. AP2 dwells in a closed, inactive state in the cytosol, but adopts an open, active conformation on the plasma membrane. Membrane-activated complexes are also phosphorylated, but the significance of this mark is debated. We recently proposed that NECAP negatively regulates AP2 by binding open and phosphorylated complexes (Beacham et al., 2018). Here, we report high-resolution cryo-EM structures of NECAP bound to phosphorylated AP2. The site of AP2 phosphorylation is directly coordinated by residues of the NECAP PHear domain that are predicted from genetic screens in C. elegans. Using membrane mimetics to generate conformationally open AP2, we find that a second domain of NECAP binds these complexes and cryo-EM reveals both domains of NECAP engaging closed, inactive AP2. Assays in vitro and in vivo confirm these domains cooperate to inactivate AP2. We propose that phosphorylation marks adaptors for inactivation.

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