Transporter tandems: precise tools for normalizing active transporter in the plasma membrane.

The transport efficiency (TE) describes the performance of a transport protein for a specific substrate. To compare the TE of different transporters, the number of active transporters in the plasma membrane must be monitored, as it may vary for each transporter and experiment. Available methods, like LC-MS quantification of tryptic peptides, fail to discriminate inactive intracellular transporters or, like cell-surface biotinylation followed by affinity chromatography and Western blotting, are imprecise and very laborious. We wanted to normalize active transporters by the activity of a second transporter. A transporter tandem, generated by joining two transporter cDNAs into a single open reading frame, should guarantee a 1 : 1 stoichiometry. Here we created a series of tandems with different linkers between the human ergothioneine (ET) transporter ETT (gene symbol SLC22A4) and organic cation transporter OCT2 (SLC22A2). The linker sequence strongly affected the expression strength. The stoichiometry was validated by absolute peptide quantification and untargeted peptide analysis. Compared with wild-type ETT, the normalized ET clearance of the natural variant L503F was higher (f = 1.34); G462E was completely inactive. The general usefulness of the tandem strategy was demonstrated by linking several transporters with ETT; every construct was active in both parts. Transporter tandems can be used - without membrane isolation or protein quantification - as precise tools for transporter number normalization, to identify, for example, relevant transporters for a drug. It is necessary, however, to find suitable linkers, to check the order of transporters, and to verify the absence of functional interference by saturation kinetics.

Knockout of the ergothioneine transporter ETT in zebrafish results in increased 8-oxoguanine levels.

Ergothioneine (ET) is a natural compound that humans and other vertebrates must absorb from dietary sources. In general, ET is considered an intracellular antioxidant. However, the precise physiological purpose of ET and the consequences of ET deficiency are still unclear. The ergothioneine transporter ETT (human gene symbol SLC22A4) is a highly specific transporter for the uptake of ET. Here, we sought to identify and knock out ETT from zebrafish (Danio rerio) to determine the function of ET. We cloned and assayed three related proteins from zebrafish, only one of which catalyzed the uptake of ET. RT-PCR analysis revealed that the protein is strongly expressed in the skin, brain, kidney, intestine, and eye. In ETT-knockout animals generated by retroviral insertion into exon 1, ET content was reduced by more than 1000-fold compared to the wild type. Thus, ETT is the sole transporter responsible for uptake of ET into zebrafish. ETT-knockout fish did not exhibit obvious differences in morphology or behavior. In whole-fish homogenates, an increase in 4-hydroxy-2,3-trans-nonenal and malondialdehyde was observed, but only after stress caused by incubation with Pb(2+) or Cu(2+). Comparison of unstressed fish at the level of small molecules by LC-MS difference shading revealed a 3.8-fold increase in 8-oxoguanine (8-oxo-7,8-dihydroguanine) in the skin of ETT-knockout animals. Our knockout represents a new model for examining the consequences of complete absence of ET. Based on the phenotype observed here, we hypothesize that the specific purpose of ET could be to eliminate singlet oxygen.

An improved procedure for subcellular spatial alignment during live-cell CLEM.

Live-cell correlative light and electron microscopy (CLEM) offers unique insights into the ultrastructure of dynamic cellular processes. A critical and technically challenging part of CLEM is the 3-dimensional relocation of the intracellular region of interest during sample processing. We have developed a simple CLEM procedure that uses toner particles from a laser printer as orientation marks. This facilitates easy tracking of a region of interest even by eye throughout the whole procedure. Combined with subcellular fluorescence markers for the plasma membrane and nucleus, the toner particles allow for precise subcellular spatial alignment of the optical and electron microscopy data sets. The toner-based reference grid is printed and transferred onto a polymer film using a standard office printer and laminator. We have also designed a polymer film holder that is compatible with most inverted microscopes, and have validated our strategy by following the ultrastructure of mitochondria that were selectively photo-irradiated during live-cell microscopy. In summary, our inexpensive and robust CLEM procedure simplifies optical imaging, without limiting the choice of optical microscope.

The protonophore CCCP interferes with lysosomal degradation of autophagic cargo in yeast and mammalian cells.

Mitophagy is a selective pathway, which targets and delivers mitochondria to the lysosomes for degradation. Depolarization of mitochondria by the protonophore CCCP is a strategy increasingly used to experimentally trigger not only mitophagy, but also bulk autophagy. Using live-cell fluorescence microscopy we found that treatment of HeLa cells with CCCP caused redistribution of mitochondrially targeted dyes, including DiOC6, TMRM, MTR, and MTG, from mitochondria to the cytosol, and subsequently to lysosomal compartments. Localization of mitochondrial dyes to lysosomal compartments was caused by retargeting of the dye, rather than delivery of mitochondrial components to the lysosome. We showed that CCCP interfered with lysosomal function and autophagosomal degradation in both yeast and mammalian cells, inhibited starvation-induced mitophagy in mammalian cells, and blocked the induction of mitophagy in yeast cells. PARK2/Parkin-expressing mammalian cells treated with CCCP have been reported to undergo high levels of mitophagy and clearance of all mitochondria during extensive treatment with CCCP. Using correlative light and electron microscopy in PARK2-expressing HeLa cells, we showed that mitochondrial remnants remained present in the cell after 24 h of CCCP treatment, although they were no longer easily identifiable as such due to morphological alterations. Our results showed that CCCP inhibits autophagy at both the initiation and lysosomal degradation stages. In addition, our data demonstrated that caution should be taken when using organelle-specific dyes in conjunction with strategies affecting membrane potential.

The serine/threonine kinase ULK1 is a target of multiple phosphorylation events.

Autophagy is a cellular degradation process that is up-regulated upon starvation. Nutrition-dependent regulation of mTOR (mammalian target of rapamycin) is a major determinant of autophagy. RTK (receptor tyrosine kinase) signalling and AMPK (AMP-activated protein kinase) converge upon mTOR to suppress or activate autophagy. Nutrition-dependent regulation of autophagy is mediated via mTOR phosphorylation of the serine/threonine kinase ULK1 (unc51-like kinase 1). In the present study, we also describe ULK1 as an mTOR-independent convergence point for AMPK and RTK signalling. We initially identified ULK1 as a 14-3-3-binding protein and this interaction was enhanced by treatment with AMPK agonists. AMPK interacted with ULK1 and phosphorylated ULK1 at Ser(555) in vitro. Mutation of this residue to alanine abrogated 14-3-3 binding to ULK1, and in vivo phosphorylation of ULK1 was blocked by a dominant-negative AMPK mutant. We next identified a high-stringency Akt site in ULK1 at Ser(774) and showed that phosphorylation at this site was increased by insulin. Finally, we found that the kinase-activation loop of ULK1 contains a consensus phosphorylation site at Thr(180) that is required for ULK1 autophosphorylation activity. Collectively, our results suggest that ULK1 may act as a major node for regulation by multiple kinases including AMPK and Akt that play both stimulatory and inhibitory roles in regulating autophagy.

Substrate discrimination by ergothioneine transporter SLC22A4 and carnitine transporter SLC22A5: gain-of-function by interchange of selected amino acids.

ETT (originally designated as OCTN1; human gene symbol SLC22A4) and CTT (OCTN2; SLC22A5) are highly specific transporters of ergothioneine and carnitine, respectively. Despite a high degree of sequence homology, both carriers discriminate precisely between substrates: ETT does not transport carnitine, and CTT does not transport ergothioneine. Our aim was to turn ETT into a transporter for carnitine and CTT into a transporter for ergothioneine by a limited number of point mutations. From a multiple alignment of several mammalian amino acid sequences, those positions were selected for conversion that were momentously different between ETT and CTT from human but conserved among all orthologues. Mutants were expressed in 293 cells and assayed for transport of ergothioneine and carnitine. Several ETT mutants clearly catalyzed transport of carnitine, up to 35% relative to wild-type CTT. Amazingly, complementary substitutions in CTT did not provoke transport activity for ergothioneine. In similar contrast, carnitine transport by CTT mutants was abolished by very few substitutions, whereas ergothioneine transport by ETT mutants was maintained even with the construct most active in carnitine transport. To explain these results, we propose that ETT and CTT use dissimilar pathways for conformational change, in addition to incongruent substrate binding sites. In other words, carnitine is excluded from ETT by binding, and ergothioneine is excluded from CTT by turnover movement. Our data indicate amino acids critical for substrate discrimination not only in transmembrane segments 5, 7, 8, and 10, but also in segments 9 and 12 which were hitherto considered as unimportant.

The carnitine transporter SLC22A5 is not a general drug transporter, but it efficiently translocates mildronate.

In addition to its function as carnitine transporter, novel organic cation transporter type 2 (OCTN2; human gene symbol SLC22A5) is widely recognized as a transporter of drugs. This notion is based on several reports of direct measurement of drug accumulation. However, a rigorous, comparative, and comprehensive analysis of transport efficiency of OCTN2 has not been available so far. In the present study, OCTN2 orthologs from human, rat, and chicken were expressed in 293 cells using an inducible expression system. Uptake of trans-4-[4-(dimethylamino)styryl]-1-methylpyridinium iodide (ASP(+)), cephaloridine, ergothioneine, gabapentin, mildronate, pyrilamine, quinidine, spironolactone, tetraethylammonium, verapamil, and vigabatrin was determined by liquid chromatography/mass spectrometry. For reference, uptake of carnitine was measured in parallel. Our results indicate that OCTN2-mediated uptake of drugs was not significantly different from zero or, with tetraethylammonium and ergothioneine, was minute relative to carnitine. The carnitine congener mildronate, by contrast, was transported very efficiently. Thus, OCTN2 is not a general drug transporter but a highly specific carrier for carnitine and closely related molecules. Transport parameters (cellular accumulation, transporter affinity, sodium dependence) were similar for mildronate and carnitine. Efficiency of transport of mildronate was even higher than that of carnitine. Hence, our results establish that OCTN2 is a key target of the cardioprotective agent mildronate because it controls, as integral protein of the plasma membrane, cellular entry of mildronate and enables efficient access to intracellular targets. The highest levels of human OCTN2 mRNA were detected by real-time reverse transcription-polymerase chain reaction in kidney, ileum, breast, small intestine, skeletal muscle, and ovary but also in some heart and central nervous system tissues.

Fast set-up of doxycycline-inducible protein expression in human cell lines with a single plasmid based on Epstein-Barr virus replication and the simple tetracycline repressor.

We have developed a novel plasmid vector, pEBTetD, for full establishment of doxycycline-inducible protein expression by just a single transfection. pEBTetD contains an Epstein-Barr virus origin of replication for stable and efficient episomal propagation in human cell lines, a cassette for continuous expression of the simple tetracycline repressor, and a cytomegalovirus-type 2 tetracycline operator (tetO2)-tetO2 promoter. As there is no integration of vector into the genome, clonal isolation of transfected cells is not necessary. Cells are thus ready for use 1 week after transfection; this contrasts with 3-12 weeks for other systems. Adequate regulation of protein expression was accomplished by abrogation of mRNA polyadenylation. In northern analysis of seven cDNAs coding for transport proteins, pools of transfected human embryonic kidney 293 cells showed on/off mRNA ratios in the order of 100:1. Cell pools were also analyzed for regulation of protein function. With two transport proteins of the plasma membrane, the on/off activity ratios were 24:1 and 34:1, respectively. With enhanced green fluorescent protein, a 23:1 ratio was observed based on fluorescence intensity data from flow cytometry. The unique advantage of our system rests on the unmodified tetracycline repressor, which is less likely, by relocation upon binding of doxycycline, to cause cellular disturbances than chimera of tetracycline repressor and eukaryotic transactivation domains. Thus, in a comprehensive comparison of on- and off-states, a steady cellular background is provided. Finally, in contrast to a system based on Flp recombinase, the set-up of our system is inherently reliable.