Cellular processing of beneficial emerging proteins.

Novel proteins can originate from non-coding DNA and contribute to species-specific adaptations. It is challenging to conceive how emerging proteins may integrate pre-existing cellular systems to bring about beneficial traits, given that their sequences are previously unseen by the cell. To address this apparent paradox, we investigated 26 emerging proteins previously associated with growth benefits in yeast.

Optimization of the fluorogen-activating protein tag for quantitative protein trafficking and co-localization studies in S. cerevisiae.

Spatial and temporal tracking of fluorescent proteins in live cells permits visualization of proteome remodeling in response to extracellular cues. Historically, protein dynamics during trafficking have been visualized using constitutively active fluorescent proteins (FPs) fused to proteins of interest. While powerful, such FPs label all cellular pools of a protein, potentially masking the dynamics of select subpopulations.

Changing course: Glucose starvation drives nuclear accumulation of Hexokinase 2 in S. cerevisiae.

Glucose is the preferred carbon source for most eukaryotes, and the first step in its metabolism is phosphorylation to glucose-6-phosphate. This reaction is catalyzed by hexokinases or glucokinases. The yeast Saccharomyces cerevisiae encodes three such enzymes, Hxk1, Hxk2, and Glk1.

A second chance at yeast early endosomes.

Post-endocytic recycling in yeast has been posited to transit solely through the Golgi, raising the possibility that yeast lack early endosomes. In this issue, Laidlaw and colleagues (2022. J.

TORC1 Signaling Controls the Stability and Function of α-Arrestins Aly1 and Aly2.

Nutrient supply dictates cell signaling changes, which in turn regulate membrane protein trafficking. To better exploit nutrients, cells relocalize membrane transporters via selective protein trafficking. Key in this reshuffling are the α-arrestins, selective protein trafficking adaptors conserved from yeast to man.

Novel mutation in hexokinase 2 confers resistance to 2-deoxyglucose by altering protein dynamics.

Glucose is central to many biological processes, serving as an energy source and a building block for biosynthesis. After glucose enters the cell, hexokinases convert it to glucose-6-phosphate (Glc-6P) for use in anaerobic fermentation, aerobic oxidative phosphorylation, and the pentose-phosphate pathway. We here describe a genetic screen in Saccharomyces cerevisiae that generated a novel spontaneous mutation in hexokinase-2, hxk2G238V, that confers resistance to the toxic glucose analog 2-deoxyglucose (2DG).

Inwardly Rectifying Potassium Channel Kir2.1 and its "Kir-ious" Regulation by Protein Trafficking and Roles in Development and Disease.

Potassium (K) homeostasis is tightly regulated for optimal cell and organismal health. Failure to control potassium balance results in disease, including cardiac arrythmias and developmental disorders. A family of inwardly rectifying potassium (Kir) channels helps cells maintain K levels.

Alpha-arrestins Aly1/Art6 and Aly2/Art3 regulate trafficking of the glycerophosphoinositol transporter Git1 and impact phospholipid homeostasis.

Background Information: Phosphatidylinositol (PI) is an essential phospholipid, critical to membrane bilayers. The complete deacylation of PI by B-type phospholipases produces intracellular and extracellular glycerophosphoinositol (GPI). Extracellular GPI is transported into the cell via Git1, a member of the Major Facilitator Superfamily of transporters at the yeast plasma membrane.

'Sugarcoating' 2-deoxyglucose: mechanisms that suppress its toxic effects.

Yeast and cancer cells are metabolically similar as they use fermentation of glucose as a primary means of generating energy. Reliance on glucose fermentation makes both of these cell types highly sensitive to the toxic glucose analog, 2-deoxyglucose. Here we review the cellular and metabolic pathways that play a role in 2-deoxyglucose sensitivity and discuss how the modifications to these pathways result in acquisition of 2-deoxyglucose resistance.

Spontaneous mutations that confer resistance to 2-deoxyglucose act through Hxk2 and Snf1 pathways to regulate gene expression and HXT endocytosis.

Yeast and fast-growing human tumor cells share metabolic similarities in that both cells use fermentation of glucose for energy and both are highly sensitive to the glucose analog 2-deoxyglucose. Spontaneous mutations in S. cerevisiae that conferred resistance to 2-deoxyglucose were identified by whole genome sequencing.

De novo emergence of adaptive membrane proteins from thymine-rich genomic sequences.

Recent evidence demonstrates that novel protein-coding genes can arise de novo from non-genic loci. This evolutionary innovation is thought to be facilitated by the pervasive translation of non-genic transcripts, which exposes a reservoir of variable polypeptides to natural selection. Here, we systematically characterize how these de novo emerging coding sequences impact fitness in budding yeast.

Helping daughters succeed: asymmetric distribution of glucose transporter mRNA.

Rapidly proliferating cells growing by glucose fermentation must first transport glucose into the cell. Both budding yeast and human tumor cells utilize members of a conserved family of glucose transporters. In this issue of , Stahl (2019) reveal that budding yeast cells confer a growth advantage to their daughters using a novel mechanism, the asymmetric distribution to the daughter cell of the mRNA for a specific glucose transporter.

AMPK-Mediated Regulation of Alpha-Arrestins and Protein Trafficking.

The adenosine monophosphate-activated protein kinase (AMPK) plays a central role in the regulation of cellular metabolism. Recent studies reveal a novel role for AMPK in the regulation of glucose and other carbohydrates flux by controlling the endocytosis of transporters. The first step in glucose metabolism is glucose uptake, a process mediated by members of the GLUT/SLC2A (glucose transporters) or HXT (hexose transporters) family of twelve-transmembrane domain glucose transporters in mammals and yeast, respectively.

Select α-arrestins control cell-surface abundance of the mammalian Kir2.1 potassium channel in a yeast model.

Protein composition at the plasma membrane is tightly regulated, with rapid protein internalization and selective targeting to the cell surface occurring in response to environmental changes. For example, ion channels are dynamically relocalized to or from the plasma membrane in response to physiological alterations, allowing cells and organisms to maintain osmotic and salt homeostasis. To identify additional factors that regulate the selective trafficking of a specific ion channel, we used a yeast model for a mammalian potassium channel, the K inward rectifying channel Kir2.

The endosomal trafficking factors CORVET and ESCRT suppress plasma membrane residence of the renal outer medullary potassium channel (ROMK).

Protein trafficking can act as the primary regulatory mechanism for ion channels with high open probabilities, such as the enal uter edullary (ROMK) channel. ROMK, also known as Kir1.1 (KCNJ1), is the major route for potassium secretion into the pro-urine and plays an indispensable role in regulating serum potassium and urinary concentrations.

Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast.

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal remodeling and vesicular trafficking in living cells. Quantitative methodologies using chimeric GFP fusions have been developed for many applications; however, GFP is somewhat resistant to proteolysis, thus its fluorescence persists in the lysosome/vacuole, which can impede quantification of cargo trafficking in the endocytic pathway. An alternative method for quantifying endocytosis and post-endocytic trafficking events makes use of superecliptic pHluorin, a pH-sensitive variant of GFP that is quenched in acidic environments.

The β subunit of yeast AMP-activated protein kinase directs substrate specificity in response to alkaline stress.

Saccharomyces cerevisiae express three isoforms of Snf1 kinase that differ by which β subunit is present, Gal83, Sip1 or Sip2. Here we investigate the abundance, activation, localization and signaling specificity of the three Snf1 isoforms. The relative abundance of these isoforms was assessed by quantitative immunoblotting using two different protein extraction methods and by fluorescence microscopy.

α-Arrestins participate in cargo selection for both clathrin-independent and clathrin-mediated endocytosis.

Clathrin-mediated endocytosis (CME) is a well-studied mechanism to internalize plasma membrane proteins; however, to endocytose such cargo, most eukaryotic cells also use alternative clathrin-independent endocytic (CIE) pathways, which are less well characterized. The budding yeast Saccharomyces cerevisiae, a widely used model for studying CME, was recently shown to have a CIE pathway that requires the GTPase Rho1, the formin Bni1, and their regulators. Nevertheless, in both yeast and mammalian cells, the mechanisms underlying cargo selection in CME and CIE are only beginning to be understood.

2-Deoxyglucose impairs Saccharomyces cerevisiae growth by stimulating Snf1-regulated and α-arrestin-mediated trafficking of hexose transporters 1 and 3.

The glucose analog 2-deoxyglucose (2DG) inhibits the growth of Saccharomyces cerevisiae and human tumor cells, but its modes of action have not been fully elucidated. Yeast cells lacking Snf1 (AMP-activated protein kinase) are hypersensitive to 2DG. Overexpression of either of two low-affinity, high-capacity glucose transporters, Hxt1 and Hxt3, suppresses the 2DG hypersensitivity of snf1Δ cells.

The proteolytic landscape of the yeast vacuole.

The vacuole in the yeast plays a number of essential roles, and to provide some of these required functions the vacuole harbors at least seven distinct proteases. These proteases exhibit a range of activities and different classifications, and they follow unique paths to arrive at their ultimate, common destination in the cell. This review will first summarize the major functions of the yeast vacuole and delineate how proteins are targeted to this organelle.

Specific α-arrestins negatively regulate Saccharomyces cerevisiae pheromone response by down-modulating the G-protein-coupled receptor Ste2.

G-protein-coupled receptors (GPCRs) are integral membrane proteins that initiate responses to extracellular stimuli by mediating ligand-dependent activation of cognate heterotrimeric G proteins. In yeast, occupancy of GPCR Ste2 by peptide pheromone α-factor initiates signaling by releasing a stimulatory Gβγ complex (Ste4-Ste18) from its inhibitory Gα subunit (Gpa1). Prolonged pathway stimulation is detrimental, and feedback mechanisms have evolved that act at the receptor level to limit the duration of signaling and stimulate recovery from pheromone-induced G1 arrest, including upregulation of the expression of an α-factor-degrading protease (Bar1), a regulator of G-protein signaling protein (Sst2) that stimulates Gpa1-GTP hydrolysis, and Gpa1 itself.

A calcineurin-dependent switch controls the trafficking function of α-arrestin Aly1/Art6.

Proper regulation of plasma membrane protein endocytosis by external stimuli is required for cell growth and survival. In yeast, excess levels of certain nutrients induce endocytosis of the cognate permeases to prevent toxic accumulation of metabolites. The α-arrestins, a family of trafficking adaptors, stimulate ubiquitin-dependent and clathrin-mediated endocytosis by interacting with both a client permease and the ubiquitin ligase Rsp5.