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The purpose of this study was to develop a method to comprehensively determine the localization of apical and basolateral membrane proteins, using a combination of apical/basolateral membrane separation and accurate SWATH (Sequential Window Acquisition of all THeoretical fragment ion spectra) proteomics. The SWATH analysis of basolateral and apical plasma membrane fractions in mouse liver quantified the protein expression of 1373 proteins. The basolateral/apical ratios of the protein expression levels were compared with the reported immunohistochemical localization for 41 model proteins (23 basolateral, 11 apical and 7 both membrane-localized proteins). Three groups were perfectly distinguished. Border lines to distinguish the apical-, both- and basolateral localizations were determined to be 0.766 and 1.42 based on probability density. The method that was established was then applied to the comprehensive determination of the proteins in mouse liver. The findings indicated that 154 and 125 proteins were localized in the apical and basolateral membranes, respectively. The levels of receptors, CD antigens and integrins, enzymes and Ras-related molecules were much higher in apical membranes than in basolateral membranes. In contrast, the levels of adhesion molecules, scaffold proteins and transporters in basolateral membranes were much higher than in apical membranes.

For more than a decade numerous evidence has been reported on the mechanisms of toxicity of α-synuclein (αS) oligomers and aggregates in α-synucleinopathies. These species were thought to form freely in the cytoplasm but recent reports of αS multimer conformations when bound to synaptic vesicles in physiological conditions, have raised the question about where αS aggregation initiates. In this review we focus on recent literature regarding the impact on membrane binding and subcellular localization of αS toxic species to understand how regular cellular function of αS contributes to pathology. Notably αS has been reported to mainly associate with specific membranes in neurons such as those of synaptic vesicles, ER/Golgi and the mitochondria, while toxic species of αS have been shown to inhibit, among others, neurotransmission, protein trafficking and mitochondrial function. Strategies interfering with αS membrane binding have shown to improve αS-driven toxicity in worms and in mice. Thus, a selective membrane binding that would result in a specific subcellular localization could be the key to understand how aggregation and pathology evolves, pointing out to αS functions that are primarily affected before onset of irreversible damage.

Morphogenesis and adaptive tropic growth in plants depend on gradients of the phytohormone auxin, mediated by the membrane-based PIN-FORMED (PIN) auxin transporters. PINs localize to a particular side of the plasma membrane (PM) or to the endoplasmic reticulum (ER) to directionally transport auxin and maintain intercellular and intracellular auxin homeostasis, respectively. However, the molecular cues that confer their diverse cellular localizations remain largely unknown. In this study, we systematically swapped the domains between ER- and PM-localized PIN proteins, as well as between apical and basal PM-localized PINs from Arabidopsis thaliana, to shed light on why PIN family members with similar topological structures reside at different membrane compartments within cells. Our results show that not only do the N- and C-terminal transmembrane domains (TMDs) and central hydrophilic loop contribute to their differential subcellular localizations and cellular polarity, but that the pairwise-matched N- and C-terminal TMDs resulting from intramolecular domain–domain coevolution are also crucial for their divergent patterns of localization. These findings illustrate the complexity of the evolutionary path of PIN proteins in acquiring their plethora of developmental functions and adaptive growth in plants.

PurposeMembrane transport protein organic anion transporting polypeptide (OATP) 1B1 mediates hepatic uptake of many drugs (e.g. statins). The OATP1B1 c.521 T > C (p. V174A) polymorphism has reduced transport activity. Conflicting in vitro results exist regarding whether V174A-OATP1B1 has reduced plasma membrane localization; no such data has been reported in physiologically relevant human liver tissue. Other potential changes, such as phosphorylation, of the V174A-OATP1B1 protein have not been explored. Current studies characterized the plasma membrane localization of V174A-OATP1B1 in genotyped human liver tissue and cell culture and compared the phosphorylation status of V174A- and wild-type (WT)-OATP1B1.MethodsLocalization of V174A- and WT-OATP1B1 were determined in OATP1B1 c.521 T > C genotyped human liver tissue (n = 79) by immunohistochemistry and in transporter-overexpressing human embryonic kidney (HEK) 293 and HeLa cells by surface biotinylation and confocal microscopy. Phosphorylation and transport of OATP1B1 was determined using 32P-orthophosphate labeling and [3H]estradiol-17β-glucuronide accumulation, respectively.ResultsAll three methods demonstrated predominant plasma membrane localization of both V174A- and WT-OATP1B1 in human liver tissue and in cell culture. Compared to WT-OATP1B1, the V174A-OATP1B1 has significantly increased phosphorylation and reduced transport.ConclusionsWe report novel findings of increased phosphorylation, but not impaired membrane localization, in association with the reduced transport function of the V174A-OATP1B1.

The Calcium-Dependent Protein Kinase (CDPK)-Related Kinase family (CRKs) consists of eight members in Arabidopsis. Recently, AtCRK5 was shown to play a direct role in the regulation of root gravitropic response involving polar auxin transport (PAT). However, limited information is available about the function of the other AtCRK genes. Here, we report a comparative analysis of the Arabidopsis CRK genes, including transcription regulation, intracellular localization, and biological function. AtCRK transcripts were detectable in all organs tested and a considerable variation in transcript levels was detected among them. Most AtCRK proteins localized at the plasma membrane as revealed by microscopic analysis of 35S::cCRK-GFP (Green Fluorescence Protein) expressing plants or protoplasts. Interestingly, 35S::cCRK1-GFP and 35S::cCRK7-GFP had a dual localization pattern which was associated with plasma membrane and endomembrane structures, as well. Analysis of T-DNA insertion mutants revealed that AtCRK genes are important for root growth and control of gravitropic responses in roots and hypocotyls. While Atcrk mutants were indistinguishable from wild type plants in short days, Atcrk1-1 mutant had serious growth defects under continuous illumination. Semi-dwarf phenotype of Atcrk1-1 was accompanied with chlorophyll depletion, disturbed photosynthesis, accumulation of singlet oxygen, and enhanced cell death in photosynthetic tissues. AtCRK1 is therefore important to maintain cellular homeostasis during continuous illumination.

Translational readthrough (TRT) of aquaporin-4 (AQP4) has remarkably expanded the importance of this new post-transcriptional mechanism, as well as the regulation potential of AQP4. The TRT isoform of AQP4, named AQP4ex, is central for both AQP4 polarization and water channel activity in the central nervous system (CNS). Here we evaluate the relevance of the TRT mechanism by analyzing whether AQP4ex is also expressed in peripheral tissues and whether the expression of AQP4ex is necessary for its polarized expression as it occurs in perivascular astrocyte processes. To this purpose, AQP4ex null mice were used, and analysis was performed by immunolocalization and immunoblot. The results demonstrate that AQP4ex is expressed in kidney, stomach, trachea and skeletal muscle with the same localization pattern as the canonical AQP4 isoforms. AQP4ex protein levels vary from 6% to about 13% of the total AQP4 protein levels in peripheral tissues. Immunogold electron microscopy experiments demonstrated the localization of AQP4ex at the astrocytic endfeet, and experiments conducted on AQP4ex null mice CNS confirmed that the expression of AQP4ex is necessary for anchoring of the perivascular AQP4. Without the readthrough isoform, AQP4 assemblies are mis-localized, being uniformly distributed on the astrocyte processes facing the neuropile. No alteration of AQP4 polarization was found in AQP4ex null kidney, stomach, trachea or skeletal muscle, suggesting that AQP4ex does not have a role for proper membrane localization of AQP4 in peripheral tissues. We conclude that a dual role for AQP4ex is limited to the CNS.

Indoleamine 2,3-dioxygenase 2 (IDO2) is a paralog of Indoleamine 2,3-dioxygenase 1 (IDO1), a tryptophan-degrading enzyme producing immunomodulatory molecules. However, the two proteins are unlikely to carry out the same functions. IDO2 shows little or no tryptophan catabolic activity and exerts contrasting immunomodulatory roles in a context-dependent manner in cancer and autoimmune diseases. The recently described potential non-enzymatic activity of IDO2 has suggested its possible involvement in alternative pathways, resulting in either pro- or anti-inflammatory effects in different models. In a previous study on non-small cell lung cancer (NSCLC) tissues, we found that IDO2 expression revealed at the plasma membrane level of tumor cells was significantly associated with poor prognosis. In this study, the A549 human cell line, basally expressing IDO2, was used as an in vitro model of human lung adenocarcinoma to gain more insights into a possible alternative function of IDO2 different from the catalytic one. In these cells, immunocytochemistry and isopycnic sucrose gradient analyses confirmed the IDO2 protein localization in the cell membrane compartment, and the immunoprecipitation of tyrosine-phosphorylated proteins revealed that kinase activities can target IDO2. The different localization from the cytosolic one and the phosphorylation state are the first indications for the signaling function of IDO2, suggesting that the IDO2 non-enzymatic role in cancer cells is worthy of deeper understanding.

RNase Y is a key endoribonuclease that regulates global mRNA turnover and processing in Bacillus subtilis and likely many other bacteria. This enzyme is anchored to the cell membrane, creating a pseudo-compartmentalization that aligns with its role in initiating the decay of mRNAs primarily translated at the cell periphery. However, the reasons behind and the consequences of RNase Y’s membrane attachment remain largely unknown. In our study, we examined a strain expressing wild-type levels of a cytoplasmic form of RNase Y from its chromosomal locus. This strain exhibits a slow-growth phenotype, similar to that of an RNase Y null mutant. Genome-wide data reveal a significant impact on the expression of hundreds of genes. While certain RNA substrates clearly depend on RNase Y’s membrane attachment, others do not. We observed no correlation between mRNA stabilization in the mutant strains and the cellular location or function of the encoded proteins. Interestingly, the Y-complex, a specificity factor for RNase Y, also appears also recognize the cytoplasmic form of the enzyme, restoring wild-type levels of the corresponding transcripts. We propose that membrane attachment of RNase Y is crucial for its functional interaction with many coding and non-coding RNAs, limiting the cleavage of specific substrates, and potentially avoiding unfavorable competition with other ribonucleases like RNase J, which shares a similar evolutionarily conserved cleavage specificity.

Human-induced pluripotent stem cells (iPS cells) are efficiently differentiated into sensory neurons. These cells express the voltage-gated sodium channel NaV1.7, which is a validated pain target. NaV1.7 deficiency leads to pain insensitivity, whereas NaV1.7 gain-of-function mutants are associated with chronic pain. During differentiation, the sensory neurons start spontaneous action potential firing around day 22, with increasing firing rate until day 40. Here, we used CRISPR/Cas9 genome editing to generate a HA-tag NaV1.7 to follow its expression during differentiation. We used two protocols to generate sensory neurons: the classical small molecule approach and a directed differentiation methodology and assessed surface NaV1.7 expression by Airyscan high-resolution microscopy. Our results show that maturation of at least 49 days is necessary to observe robust NaV1.7 surface expression in both protocols. Electric activity of the sensory neurons precedes NaV1.7 surface expression. A clinically effective NaV1.7 blocker is still missing, and we expect this iPS cell model system to be useful for drug discovery and disease modeling.

Candidiasis, which presents a substantial risk to human well‐being, is frequently treated with azoles. However, drug‐drug interactions caused by azoles inhibiting the human CYP3A4 enzyme, together with increasing resistance of Candida species to azoles, represent serious issues with this class of drug, making it imperative to develop innovative antifungal drugs to tackle this growing clinical challenge. A drug repurposing approach is used to examine a library of Food and Drug Administration (FDA)‐approved drugs, ultimately identifying otilonium bromide (OTB) as an exceptionally encouraging antifungal agent. Mechanistically, OTB impairs vesicle‐mediated trafficking by targeting Sec31, thereby impeding the plasma membrane (PM) localization of the ergosterol transporters, such as Sip3. Consequently, OTB obstructs the movement of ergosterol across membranes and triggers cytotoxic autophagy. It is noteworthy that C. albicans encounters challenges in developing resistance to OTB because it is not a substrate for drug transporters. This study opens a new door for antifungal therapy, wherein OTB disrupts ergosterol subcellular distribution and induces cytotoxic autophagy. Additionally, it circumvents the hepatotoxicity associated with azole‐mediated liver enzyme inhibition and avoids export‐mediated drug resistance in C. albicans. Otilonium bromide (OTB) demonstrates efficacy against Candida albicans by targeting Sec31, thereby impeding vesicle transport and ergosterol localization, ultimately inducing cytotoxic autophagy. C. albicans exhibits susceptibility to OTB because it does not act as a substrate of drug pumps. The mechanism of action of OTB, involving modulation of ergosterol distribution and initiation of autophagy, offers a novel antifungal strategy.