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. Internalized GPI is degraded to produce inositol, phosphate and glycerol, thereby contributing to these pools. GIT1 gene expression is controlled by nutrient balance, with phosphate or inositol starvation increasing GIT1 expression to stimulate GPI uptake. However, less is known about control of Git1 protein levels or localization. RESULTS: We find that the α-arrestins, an important class of protein trafficking adaptor, regulate Git1 localization and this is dependent upon their interaction with the ubiquitin ligase Rsp5. Specifically, α-arrestin Aly2 stimulates Git1 trafficking to the vacuole under basal conditions, but in response to GPI-treatment, either Aly1 or Aly2 promote Git1 vacuole trafficking. Cell surface retention of Git1, as occurs in aly1∆ aly2∆ cells, is linked to impaired growth in the presence of exogenous GPI and results in increased uptake of radiolabeled GPI, suggesting that accumulation of GPI somehow causes cellular toxicity. Regulation of α-arrestin Aly1 by the protein phosphatase calcineurin improves steady-state and substrate-induced trafficking of Git1, however, calcineurin plays a larger role in Git1 trafficking beyond regulation of α-arrestins. Interestingly, loss of Aly1 and Aly2 increased phosphatidylinositol-3-phosphate on the limiting membrane of the vacuole, and this was further exacerbated by GPI addition, suggesting that the effect is partially linked to Git1. Loss of Aly1 and Aly2 leads to increased incorporation of inositol label from [3 H]-inositol-labelled GPI into PI, confirming that internalized GPI influences PI balance and indicating a role for the a-arrestins in this regulation. CONCLUSIONS: The α-arrestins Aly1 and Aly2 are novel regulators of Git1 trafficking with previously unanticipated roles in controlling phospholipid distribution and balance. SIGNIFICANCE: To our knowledge, this is the first example of α-arrestin regulation of phosphatidyliniositol-3-phosphate levels. In future studies it will be exciting to determine if other α-arrestins similarly alter PI and PIPs to change the cellular landscape.

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

Spatial and temporal tracking of fluorescent proteins (FPs) in live cells permits visualization of proteome remodeling in response to extracellular cues. Historically, protein dynamics during trafficking have been visualized using constitutively active FPs fused to proteins of interest. While powerful, such FPs label all cellular pools of a protein, potentially masking the dynamics of select subpopulations. To help study protein subpopulations, bioconjugate tags, including the fluorogen activation proteins (FAPs), were developed. FAPs are comprised of two components: a single-chain antibody (SCA) fused to the protein of interest and a malachite-green (MG) derivative, which fluoresces only when bound to the SCA. Importantly, the MG derivatives can be either cell-permeant or -impermeant, thus permitting isolated detection of SCA-tagged proteins at the cell surface and facilitating quantitative endocytic measures. To expand FAP use in yeast, we optimized the SCA for yeast expression, created FAP-tagging plasmids, and generated FAP-tagged organelle markers. To demonstrate FAP efficacy, we coupled the SCA to the yeast G-protein coupled receptor Ste3. We measured Ste3 endocytic dynamics in response to pheromone and characterized cis- and trans-acting regulators of Ste3. Our work significantly expands FAP technology for varied applications in S. cerevisiae.

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. To help study protein subpopulations, bioconjugate tags, including the fluorogen activation proteins (FAPs), were developed. FAPs are comprised of two components: a single-chain antibody (SCA) fused to the protein of interest and a malachite-green (MG) derivative, which fluoresces only when bound to the SCA. Importantly, the MG derivatives can be either cell-permeant or -impermeant, thus permitting isolated detection of SCA-tagged proteins at the cell surface and facilitating quantitative endocytic measures. To expand FAP use in yeast, we optimized the SCA for yeast expression, created FAP-tagging plasmids, and generated FAP-tagged organelle markers. To demonstrate FAP efficacy, we coupled the SCA to the yeast G-protein coupled receptor Ste3. We measured Ste3 endocytic dynamics in response to pheromone and characterized cis- and trans-acting regulators of Ste3. Our work significantly expands FAP technology for varied applications in S. cerevisiae.

Phase separation in immune signalling.

Immune signalling pathways convert pathogenic stimuli into cytosolic events that lead to the resolution of infection. Upon ligand engagement, immune receptors together with their downstream adaptors and effectors undergo substantial conformational changes and spatial reorganization. During this process, nanometre-to-micrometre-sized signalling clusters have been commonly observed that are believed to be hotspots for signal transduction. Because of their large size and heterogeneous composition, it remains a challenge to fully understand the mechanisms by which these signalling clusters form and their functional consequences. Recently, phase separation has emerged as a new biophysical principle for organizing biomolecules into large clusters with fluidic properties. Although the field is still in its infancy, studies of phase separation in immunology are expected to provide new perspectives for understanding immune responses. Here, we present an up-to-date view of how liquid-liquid phase separation drives the formation of signalling condensates and regulates immune signalling pathways, including those downstream of T cell receptor, B cell receptor and the innate immune receptors cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) and retinoic acid-inducible gene I protein (RIG-I). We conclude with a summary of the current challenges the field is facing and outstanding questions for future studies.

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. Encoded by KCNJ genes, Kir channels are comprised of a tetramer of Kir subunits, each of which contains two-transmembrane domains. The assembled Kir channel generates an ion selectivity filter for K+ at the monomer interface, which allows for K+ transit. Kir channels are found in many cell types and influence K+ homeostasis across the organism, impacting muscle, nerve and immune function. Kir2.1 is one of the best studied family members with well-defined roles in regulating heart rhythm, muscle contraction and bone development. Due to their expansive roles, it is not surprising that Kir mutations lead to disease, including cardiomyopathies, and neurological and metabolic disorders. Kir malfunction is linked to developmental defects, including underdeveloped skeletal systems and cerebellar abnormalities. Mutations in Kir2.1 cause the periodic paralysis, cardiac arrythmia, and developmental deficits associated with Andersen-Tawil Syndrome. Here we review the roles of Kir family member Kir2.1 in maintaining K+ balance with a specific focus on our understanding of Kir2.1 channel trafficking and emerging roles in development and disease. We provide a synopsis of the vital work focused on understanding the trafficking of Kir2.1 and its role in development.