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 Kir2.1. Kir2.1 maintains potassium homeostasis in heart muscle cells, and Kir2.1 defects lead to human disease. By examining the ability of Kir2.1 to rescue the growth of yeast cells lacking endogenous potassium channels, we discovered that specific α-arrestins regulate Kir2.1 localization. Specifically, we found that the Ldb19/Art1, Aly1/Art6, and Aly2/Art3 α-arrestin adaptor proteins promote Kir2.1 trafficking to the cell surface, increase Kir2.1 activity at the plasma membrane, and raise intracellular potassium levels. To better quantify the intracellular and cell-surface populations of Kir2.1, we created fluorogen-activating protein fusions and for the first time used this technique to measure the cell-surface residency of a plasma membrane protein in yeast. Our experiments revealed that two α-arrestin effectors also control Kir2.1 localization. In particular, both the Rsp5 ubiquitin ligase and the protein phosphatase calcineurin facilitated the α-arrestin-mediated trafficking of Kir2.1. Together, our findings implicate α-arrestins in regulating an additional class of plasma membrane proteins and establish a new tool for dissecting the trafficking itinerary of any membrane protein in yeast.

The role of the subchondral layer in osteonecrosis of the femoral head: analysis based on HR-QCT in comparison to MRI findings.

BACKGROUND: Non-traumatic avascular osteonecrosis of the femoral head (ONFH) is a severe disease causing destruction of the hip joint, often necessitating total hip arthroplasty (THA) even in young patients. Magnetic resonance imaging (MRI) is commonly used for diagnosis of ONFH, but provides limited insight into the subchondral bone microstructure. PURPOSE: To analyze routine MRI findings in comparison to high-resolution quantitative computed tomography (HR-QCT) with a special focus on the subchondral layer and to estimate the importance of differences determining the indication for THA. MATERIAL AND METHODS: Twelve patients with ONFH were included before THA. Preoperative MRI and HR-QCT of the retrieved femoral heads were aligned using a registration algorithm. Pathological findings and trabecular bone parameters in matched areas were analyzed by two readers. McNemar, marginal homogeneity test, and Pearson's correlation coefficient were used for comparison. RESULTS: Subchondral delamination was found in nine cases on HR-QCT, but missed or underestimated in all but one case on MRI ( P = 0.016). Chondral discontinuity was found in all cases on HR-QCT and in two cases on MRI ( P = 0.016). Areas of complete bone resorption on HR-QCT were linked to high signal intensity on 3D gradient-echo MRI sequences with water-selective excitation, while there was no correlation between trabecular bone parameters and MRI signal intensities in other areas ( P = 0.304). CONCLUSION: Subchondral delamination, subchondral resorption, and chondral discontinuity are found frequently in advanced stages of ONFH. These lesions tend to be underestimated on conventional MRI. Our results support the importance of CT imaging in the evaluation of ONFH.

Saccharomyces cerivisiae as a model system for kidney disease: what can yeast tell us about renal function?

Ion channels, solute transporters, aquaporins, and factors required for signal transduction are vital for kidney function. Because mutations in these proteins or in associated regulatory factors can lead to disease, an investigation into their biogenesis, activities, and interplay with other proteins is essential. To this end, the yeast, Saccharomyces cerevisiae, represents a powerful experimental system. Proteins expressed in yeast include the following: 1) ion channels, including the epithelial sodium channel, members of the inward rectifying potassium channel family, and cystic fibrosis transmembrane conductance regulator; 2) plasma membrane transporters, such as the Na(+)-K(+)-ATPase, the Na(+)-phosphate cotransporter, and the Na(+)-H(+) ATPase; 3) aquaporins 1-4; and 4) proteins such as serum/glucocorticoid-induced kinase 1, phosphoinositide-dependent kinase 1, Rh glycoprotein kidney, and trehalase. The variety of proteins expressed and studied emphasizes the versatility of yeast, and, because of the many available tools in this organism, results can be obtained rapidly and economically. In most cases, data gathered using yeast have been substantiated in higher cell types. These attributes validate yeast as a model system to explore renal physiology and suggest that research initiated using this system may lead to novel therapeutics.

ESCRT regulates surface expression of the Kir2.1 potassium channel.

Protein quality control (PQC) is required to ensure cellular health. PQC is recognized for targeting the destruction of defective polypeptides, whereas regulated protein degradation mechanisms modulate the concentration of specific proteins in concert with physiological demands. For example, ion channel levels are physiologically regulated within tight limits, but a system-wide approach to define which degradative systems are involved is lacking. We focus on the Kir2.1 potassium channel because altered Kir2.1 levels lead to human disease and Kir2.1 restores growth on low-potassium medium in yeast mutated for endogenous potassium channels. Using this system, first we find that Kir2.1 is targeted for endoplasmic reticulum-associated degradation (ERAD). Next a synthetic gene array identifies nonessential genes that negatively regulate Kir2.1. The most prominent gene family that emerges from this effort encodes members of endosomal sorting complex required for transport (ESCRT). ERAD and ESCRT also mediate Kir2.1 degradation in human cells, with ESCRT playing a more prominent role. Thus multiple proteolytic pathways control Kir2.1 levels at the plasma membrane.

The endoplasmic reticulum-associated degradation of the epithelial sodium channel requires a unique complement of molecular chaperones.

The epithelial sodium channel (ENaC) is composed of a single copy of an alpha-, beta-, and gamma-subunit and plays an essential role in water and salt balance. Because ENaC assembles inefficiently after its insertion into the ER, a substantial percentage of each subunit is targeted for ER-associated degradation (ERAD). To define how the ENaC subunits are selected for degradation, we developed novel yeast expression systems for each ENaC subunit. Data from this analysis suggested that ENaC subunits display folding defects in more than one compartment and that subunit turnover might require a unique group of factors. Consistent with this hypothesis, yeast lacking the lumenal Hsp40s, Jem1 and Scj1, exhibited defects in ENaC degradation, whereas BiP function was dispensable. We also discovered that Jem1 and Scj1 assist in ENaC ubiquitination, and overexpression of ERdj3 and ERdj4, two lumenal mammalian Hsp40s, increased the proteasome-mediated degradation of ENaC in vertebrate cells. Our data indicate that Hsp40s can act independently of Hsp70 to select substrates for ERAD.