Multispanning Membrane Protein Research Articles

From bacteria to chloroplasts: evolution of the chloroplast SRP system.

Chloroplasts derive from a prokaryotic symbiont that lost most of its genes during evolution. As a result, the great majority of chloroplast proteins are encoded in the nucleus and are posttranslationally imported into the organelle. The chloroplast genome encodes only a few proteins. These include several multispan thylakoid membrane proteins which are synthesized on thylakoid-bound ribosomes and cotranslationally inserted into the membrane. During evolution, ancient prokaryotic targeting machineries were adapted and combined with novel targeting mechanisms to facilitate post- and cotranslational protein transport in chloroplasts. This review focusses on the chloroplast signal recognition particle (cpSRP) protein transport system, which has been intensively studied in higher plants. The cpSRP system derived from the prokaryotic SRP pathway, which mediates the cotranslational protein transport to the bacterial plasma membrane. Chloroplasts contain homologs of several components of the bacterial SRP system. The function of these conserved components in post- and/or cotranslational protein transport and chloroplast-specific modifications of these transport mechanisms are described. Furthermore, recent studies of cpSRP systems in algae and lower plants are summarized and their impact on understanding the evolution of the cpSRP system are discussed.

Downregulation of LAPTM5 suppresses cell proliferation and viability inducing cell cycle arrest at G0/G1 phase of bladder cancer cells.

Our transcriptome analysis revealed in bladder cancer (BCa) tissues a significant induction of lysosomal-associated multispanning membrane protein5 (LAPTM5), a lysosomal membrane protein preferentially expressing in immune cells and hematopoietic cells. Transportation of LAPTM5 from Golgi to lysosome could be inhibited by deficiency of Nedd4, a key member of E3 ubiquitin ligase family overexpressing in invasive BCa and promoting its progression. Therefore, we hypothesize that LAPTM5 may be closely correlated with BCa tumorigenesis. In human BCa tissues, we observed that LAPTM5 was significantly induced at both mRNA and protein levels, which is consistent with our microarray result. Furthermore, we established a BCa cell model with downregulated LAPTM5, revealing a significantly delayed growth rate in the BCa cells with knockdown of LAPTM5. Moreover, cell cycle arrest at G0/G1 phase was triggered by decreased LAPTM5 as well, which could lead to delayed BCa cell growth. In contrast, no significant alteration of apoptosis in the BCa cells with downregulated LAPTM5 was noticed. Analysis of the changes of migration and invasion, showed significant reduced LAPTM5 suppressed cell metastasis. Furthermore, proteins involved in epithelial-mesenchymal transition (EMT) were strongly altered, which plays a central role in metastasis. In addition, phosphorylated ERK1/2 and p38, key members of mitogen-activated protein kinase (MAPK) family regulating BCa tumorigenesis, were strongly decreased. Taken together, our results suggested that decreased LAPTM5 inhibited proliferation and viability, as well as induced G0/G1 cell cycle arrest possibly via deactivation of ERK1/2 and p38 in BCa cells.

Global profiling of SRP interaction with nascent polypeptides.

Signal recognition particle (SRP) is a universally conserved protein-RNA complex that mediates co-translational protein translocation and membrane insertion by targeting translating ribosomes to membrane translocons. The existence of parallel co- and post-translational transport pathways, however, raises the question of the cellular substrate pool of SRP and the molecular basis of substrate selection. Here we determine the binding sites of bacterial SRP within the nascent proteome of Escherichia coli at amino acid resolution, by sequencing messenger RNA footprints of ribosome-nascent-chain complexes associated with SRP. SRP, on the basis of its strong preference for hydrophobic transmembrane domains (TMDs), constitutes a compartment-specific targeting factor for nascent inner membrane proteins (IMPs) that efficiently excludes signal-sequence-containing precursors of periplasmic and outer membrane proteins. SRP associates with hydrophobic TMDs enriched in consecutive stretches of hydrophobic and bulky aromatic amino acids immediately on their emergence from the ribosomal exit tunnel. By contrast with current models, N-terminal TMDs are frequently skipped and TMDs internal to the polypeptide sequence are selectively recognized. Furthermore, SRP binds several TMDs in many multi-spanning membrane proteins, suggesting cycles of SRP-mediated membrane targeting. SRP-mediated targeting is not accompanied by a transient slowdown of translation and is not influenced by the ribosome-associated chaperone trigger factor (TF), which has a distinct substrate pool and acts at different stages during translation. Overall, our proteome-wide data set of SRP-binding sites reveals the underlying principles of pathway decisions for nascent chains in bacteria, with SRP acting as the dominant triaging factor, sufficient to separate IMPs from substrates of the SecA-SecB post-translational translocation and TF-assisted cytosolic protein folding pathways.

Chemogenetic E-MAP in Saccharomyces cerevisiae for Identification of Membrane Transporters Operating Lipid Flip Flop.

While most yeast enzymes for the biosynthesis of glycerophospholipids, sphingolipids and ergosterol are known, genes for several postulated transporters allowing the flopping of biosynthetic intermediates and newly made lipids from the cytosolic to the lumenal side of the membrane are still not identified. An E-MAP measuring the growth of 142'108 double mutants generated by systematically crossing 543 hypomorphic or deletion alleles in genes encoding multispan membrane proteins, both on media with or without an inhibitor of fatty acid synthesis, was generated. Flc proteins, represented by 4 homologous genes encoding presumed FAD or calcium transporters of the ER, have a severe depression of sphingolipid biosynthesis and elevated detergent sensitivity of the ER. FLC1, FLC2 and FLC3 are redundant in granting a common function, which remains essential even when the severe cell wall defect of flc mutants is compensated by osmotic support. Biochemical characterization of some other genetic interactions shows that Cst26 is the enzyme mainly responsible for the introduction of saturated very long chain fatty acids into phosphatidylinositol and that the GPI lipid remodelase Cwh43, responsible for introducing ceramides into GPI anchors having a C26:0 fatty acid in sn-2 of the glycerol moiety can also use lyso-GPI protein anchors and various base resistant lipids as substrates. Furthermore, we observe that adjacent deletions in several chromosomal regions show strong negative genetic interactions with a single gene on another chromosome suggesting the presence of undeclared suppressor mutations in certain chromosomal regions that need to be identified in order to yield meaningful E-map data.

Molecular determinants that mediate the sorting of human ATG9A from the endoplasmic reticulum

ATG9A is a multispanning membrane protein required for autophagosome formation. Under basal conditions, neosynthesized ATG9A proteins travel to the Golgi apparatus and cycle between the trans-Golgi network and endosomes. In the present work, we searched for molecular determinants involved in the subcellular trafficking of human ATG9A in HeLa cells using sequential deletions and point mutations. Deletion of amino acids L340 to L354 resulted in the retention of ATG9A in the endoplasmic reticulum. In addition, we found that substitution of the L711YM713 sequence (located in the C-terminal region of ATG9A) by alanine residues severely impaired its transport through the Golgi apparatus. This defect could be corrected by oligomerization of the mutant protein with co-transfected wild-type ATG9A, suggesting that ATG9A oligomerization may help its sorting through biosynthetic compartments. Lastly, the study of the consequences of the LYM/AAA mutation on the intracellular trafficking of ATG9A highlighted that some newly synthesized ATG9A can bypass the Golgi apparatus to reach the plasma membrane. Taken together, these findings provide new insights into the intracellular pathways followed by ATG9A to reach different subcellular compartments, and into the intramolecular determinants that drive the sorting of this protein.

TRAM1 protects AR42J cells from caerulein-induced acute pancreatitis through ER stress-apoptosis pathway

Chronic endoplasmic reticulum (ER) stress in pancreatic acinar cells has emerged as a major contributor to the recovery of acute pancreatitis (AP). However, the molecular mechanisms linking AP and ER stress remain not fully understood. In this study, we employed caerulein to induce AP-like inflammation in the AR42J rat pancreatic acinar cells to mimic the AP-like acinar cell injury. Caerulein can activate ER stress in AR42J cells, but the molecular link between AP and ER stress remains to be identified. We here reported that translocating chain-associated membrane protein 1 (TRAM1), an ER-resident multispanning membrane protein, was involved in the onset of AP-like injury on AR42J cells. TRAM1 was significantly elevated in caerulein-treated AR42J cells. Furthermore, we showed that knockdown of TRAM1 led to hyperactivation of 78kDa glucose-regulated protein precursor (GRP78) and C/EBP homologous protein (CHOP) and the activation of downstream apoptosis pathway. Given the fact that the activation of ER stress played a protection role in AP, the pro-inflammatory mediators TNF-α and IL-6 and the marker of cell injury LDH were also analyzed. We found that depletion of TRAM1 markedly increased the secretion of TNF-α, IL-6, and LDH in the cells. Moreover, flow cytometry indicated that treatment with caerulein induced a significant decrease of apoptotic index and increase of necrosis index in TRAM1-siRNA cells, compared with control groups, as indicated by downregulated expression of cleaved caspase-3, caspase-8, and caspase-9 mRNA expression activity in TRAM1-siRNA cells. These data implicated that TRAM1 might protect AR42J cells against caerulein-induced AP in AR42J cells through alleviating ER stress.

The ssrA-Tag Facilitated Degradation of an Integral Membrane Protein.

ATP-dependent degradation plays a critical role in the quality control and recycling of proteins in cells. However, complete degradation of membrane proteins by ATP-dependent proteases in bacteria is not well-studied. We discovered that the degradation of a multidomain and multispan integral membrane protein AcrB could be facilitated by the introduction of a ssrA-tag at the C-terminus of the protein sequence and demonstrated that the cytoplasmic unfoldase-protease complex ClpXP was involved in the degradation. This is the first report to our knowledge to reveal that the ClpXP complex is capable of degrading integral membrane proteins. The chaperone SspB also played a role in the degradation. Using purified proteins, we demonstrated that the addition of the ssrA-tag did not drastically affect the structure of AcrB, and the degradation of detergent solubilized AcrB by purified ClpXP could be observed in vitro.

Accurate Prediction of Contact Numbers for Multi-Spanning Helical Membrane Proteins.

Prediction of the three-dimensional (3D) structures of proteins by computational methods is acknowledged as an unsolved problem. Accurate prediction of important structural characteristics such as contact number is expected to accelerate the otherwise slow progress being made in the prediction of 3D structure of proteins. Here, we present a dropout neural network-based method, TMH-Expo, for predicting the contact number of transmembrane helix (TMH) residues from sequence. Neuronal dropout is a strategy where certain neurons of the network are excluded from back-propagation to prevent co-adaptation of hidden-layer neurons. By using neuronal dropout, overfitting was significantly reduced and performance was noticeably improved. For multi-spanning helical membrane proteins, TMH-Expo achieved a remarkable Pearson correlation coefficient of 0.69 between predicted and experimental values and a mean absolute error of only 1.68. In addition, among those membrane protein-membrane protein interface residues, 76.8% were correctly predicted. Mapping of predicted contact numbers onto structures indicates that contact numbers predicted by TMH-Expo reflect the exposure patterns of TMHs and reveal membrane protein-membrane protein interfaces, reinforcing the potential of predicted contact numbers to be used as restraints for 3D structure prediction and protein-protein docking. TMH-Expo can be accessed via a Web server at www.meilerlab.org .

Determinants of Multispanning Membrane Protein Integration Mediated by the Sec Translocon

Multispanning membrane proteins - i.e., proteins with multiple transmembrane domains that thread back-and-forth across the cell membrane - are essential for cellular functions that include signal transduction, material transport, and energy conversion. Performing these functions requires the proteins to integrate within the membrane in the correct topology, or the overall orientation of the protein relative to the membrane. The integration of most proteins proceeds via the Sec translocon, a conserved protein-conducting channel that allows a nascent protein chain to access the membrane while being fed into the channel during translation. Previous studies have established that the Sec-facilitated integration of single-spanning proteins depends on factors including nascent chain hydrophobicity and charge; however, the extent to which these properties influence the integration and topology of multispanning proteins is less clear. Here, we use coarse-grained simulations to investigate the Sec-facilitated membrane integration of multispanning proteins on realistic biological timescales. We employ a simulation model that enables access to a timescale of minutes while retaining sufficient chemical accuracy to capture the forces that drive membrane integration. This study focuses on understanding two experimental observations: first, the finding that the protein EmrE exhibits two possible topologies in a 1:1 stoichiometry; and second, the finding that some marginally hydrophobic domains efficiently integrate into the membrane as part of a multispanning protein, despite inefficiently integrating as a single-spanning protein. This work provides mechanistic explanations for these observations, leading to insight into the sequence-level determinants of multispanning membrane protein integration and topology (1). (1) R.C. Van Lehn, B. Zhang, and T. F. Miller III, “Regulation of multispanning membrane protein topology by post-translational annealing,” eLife, in press.

Stability and flexibility of marginally hydrophobic-segment stalling at the endoplasmic reticulum translocon.

Many membrane proteins are integrated into the endoplasmic reticulum membrane through the protein-conducting channel, the translocon. Transmembrane segments with insufficient hydrophobicity for membrane integration are frequently found in multispanning membrane proteins, and such marginally hydrophobic (mH) segments should be accommodated, at least transiently, at the membrane. Here we investigated how mH-segments stall at the membrane and their stability. Our findings show that mH-segments can be retained at the membrane without moving into the lipid phase and that such segments flank Sec61α, the core channel of the translocon, in the translational intermediate state. The mH-segments are gradually transferred from the Sec61 channel to the lipid environment in a hydrophobicity-dependent manner, and this lateral movement may be affected by the ribosome. In addition, stalling mH-segments allow for insertion of the following transmembrane segment, forming an Ncytosol/Clumen orientation, suggesting that mH-segments can move laterally to accommodate the next transmembrane segment. These findings suggest that mH-segments may be accommodated at the ER membrane with lateral fluctuation between the Sec61 channel and the lipid phase.

Capsid protease domain as a tool for assessing protein-domain folding during organelle import of nascent polypeptides in living cells.

Various proteins synthesized by ribosomes are imported into specific organelles. To elucidate the behavior of protein domains during import, we developed a folding probe, in which the capsid protease (CP) domain of the Semliki Forest virus was connected to enhanced green fluorescent protein (EGFP). The probe was fused to appropriate N-terminal organelle-targeting signal sequences and expressed in cultured cells. When the entire CP-domain was present in the cytosol, it became folded and cleaved off the following EGFP-domain. Once cleaved, EGFP stability was not affected by upstream sequences. Based on EGFP localization, we estimated the extent of CP-domain folding in the cytosolic space. When fused to mitochondrial hydrophobic multispanning membrane protein ABCB10, more than half of the EGFP remained in the cytoplasm, whereas most of the CP-portion was in the mitochondrial fraction. When fused to the endoplasmic reticulum (ER) signal, the cleaved EGFP was observed only in the ER fraction, confirming that the CP-domain cannot fold on the cytoplasmic side during cotranslational ER translocation. Thus, import of the ABCB10 molecule was not as tightly coupled with chain elongation as ER translocation. Use of this probe to quantitatively examine stop-translocation at the ER translocon in living cells revealed that positively charged residues on the translocating nascent chain stall at the ER translocon.

Regulation of multispanning membrane protein topology via post-translational annealing.

The canonical mechanism for multispanning membrane protein topogenesis suggests that protein topology is established during cotranslational membrane integration. However, this mechanism is inconsistent with the behavior of EmrE, a dual-topology protein for which the mutation of positively charged loop residues, even close to the C-terminus, leads to dramatic shifts in its topology. We use coarse-grained simulations to investigate the Sec-facilitated membrane integration of EmrE and its mutants on realistic biological timescales. This work reveals a mechanism for regulating membrane-protein topogenesis, in which initially misintegrated configurations of the proteins undergo post-translational annealing to reach fully integrated multispanning topologies. The energetic barriers associated with this post-translational annealing process enforce kinetic pathways that dictate the topology of the fully integrated proteins. The proposed mechanism agrees well with the experimentally observed features of EmrE topogenesis and provides a range of experimentally testable predictions regarding the effect of translocon mutations on membrane protein topogenesis.

Glycosylation of solute carriers: mechanisms and functional consequences.

Solute carriers (SLCs) are one of the largest groups of multi-spanning membrane proteins in mammals and include ubiquitously expressed proteins as well as proteins with highly restricted tissue expression. A vast number of studies have addressed the function and organization of SLCs as well as their posttranslational regulation, but only relatively little is known about the role of SLC glycosylation. Glycosylation is one of the most abundant posttranslational modifications of animal proteins and through recent advances in our understanding of protein-glycan interactions, the functional roles of SLC glycosylation are slowly emerging. The purpose of this review is to provide a concise overview of the aspects of glycobiology most relevant to SLCs, to discuss the roles of glycosylation in the regulation and function of SLCs, and to outline the major open questions in this field, which can now be addressed given major technical advances in this and related fields of study in recent years.

Multiple acquisition of magic angle spinning solid-state NMR experiments using one receiver: Application to microcrystalline and membrane protein preparations

Solid-state NMR spectroscopy of proteins is a notoriously low-throughput technique. Relatively low-sensitivity and poor resolution of protein samples require long acquisition times for multidimensional NMR experiments. To speed up data acquisition, we developed a family of experiments called Polarization Optimized Experiments (POE), in which we utilized the orphan spin operators that are discarded in classical multidimensional NMR experiments, recovering them to allow simultaneous acquisition of multiple 2D and 3D experiments, all while using conventional probes with spectrometers equipped with one receiver. POE allow the concatenation of multiple 2D or 3D pulse sequences into a single experiment, thus potentially combining all of the aforementioned advances, boosting the capability of ssNMR spectrometers at least two-fold without the addition of any hardware. In this perspective, we describe the first generation of POE, such as dual acquisition MAS (or DUMAS) methods, and then illustrate the evolution of these experiments into MEIOSIS, a method that enables the simultaneous acquisition of multiple 2D and 3D spectra. Using these new pulse schemes for the solid-state NMR investigation of biopolymers makes it possible to obtain sequential resonance assignments, as well as distance restraints, in about half the experimental time. While designed for acquisition of heteronuclei, these new experiments can be easily implemented for proton detection and coupled with other recent advancements, such as dynamic nuclear polarization (DNP), to improve signal to noise. Finally, we illustrate the application of these methods to microcrystalline protein preparations as well as single and multi-span membrane proteins reconstituted in lipid membranes.

Vmp1 regulates PtdIns3P signaling during autophagosome formation in Dictyostelium discoideum.

Generation and turnover of phosphatidylinositol 3-phosphate (PtdIns3P) signaling is essential for autophagosome formation and other membrane traffic processes. In both Dictyostelium discoideum and mammalian cells, autophagosomes are formed from specialized regions of the endoplasmic reticulum (ER), called omegasomes, which are enriched in the signaling lipid PtdIns3P. Vacuole membrane protein 1 (Vmp1) is a multispanning membrane protein localized at the ER that is required for autophagosome formation. There are conflicting reports in the literature as to whether Vmp1 is strictly required or not for autophagy-related PtdIns3P signaling and its hierarchical relationship with Atg1 and PI3K. We have now addressed these questions in the Dictyostelium model. We show that Dictyostelium cells lacking Vmp1 have elevated and aberrant PtdIns3P signaling on the ER, resulting in an increased and persistent recruitment of Atg18 and other autophagic proteins. This indicates that Vmp1 is not strictly essential for the generation of PtdIns3P signaling but rather suggests a role in the correct turnover or modulation of this signaling. Of interest, these PtdIns3P-enriched regions of the ER surround ubiquitinated protein aggregates but are unable to form functional autophagosomes. vmp1 null cells also have additional defects in macropinocytosis and growth, which are not shared by other autophagy mutants. Remarkably, we show that these defects and also the aberrant PtdIns3P distribution are largely suppressed by the concomitant loss of Atg1, indicating that aberrant autophagic signaling on the ER inhibits macropinocytosis. These results suggest that Atg1 functions upstream of Vmp1 in this signaling pathway and demonstrates a previously unappreciated link between abnormal autophagy signaling and macropinocytosis.

Mode of membrane insertion of individual transmembrane segments in Mdl1 and Mdl2, multi-spanning mitochondrial ABC transporters

The sorting of an individual transmembrane (TM) segment of multi-spanning membrane proteins by the TIM23 complex in the mitochondrial inner membrane is poorly understood. Using the Mgm1 fusion approach, we attempted to assess the membrane insertion of individual TM segments of Mdl1p and Mdl2p, mitochondrial ABC transporters. Although these transporters share high sequence similarity, our results show that their membrane sorting patterns differ and that specific residues in TM domains strongly influence membrane insertion or translocation. These data imply that TIM23-mediated membrane insertion highly depends on the TM domain sequence context.

The Positive Inside Rule Is Stronger When Followed by a Transmembrane Helix

The translocon recognizes transmembrane helices with sufficient level of hydrophobicity and inserts them into the membrane. However, sometimes less hydrophobic helices are also recognized. Positive inside rule, orientational preferences of and specific interactions with neighboring helices have been shown to aid in the recognition of these helices, at least in artificial systems. To better understand how the translocon inserts marginally hydrophobic helices, we studied three naturally occurring marginally hydrophobic helices, which were previously shown to require the subsequent helix for efficient translocon recognition. We find no evidence for specific interactions when we scan all residues in the subsequent helices. Instead, we identify arginines located at the N-terminal part of the subsequent helices that are crucial for the recognition of the marginally hydrophobic transmembrane helices, indicating that the positive inside rule is important. However, in two of the constructs, these arginines do not aid in the recognition without the rest of the subsequent helix; that is, the positive inside rule alone is not sufficient. Instead, the improved recognition of marginally hydrophobic helices can here be explained as follows: the positive inside rule provides an orientational preference of the subsequent helix, which in turn allows the marginally hydrophobic helix to be inserted; that is, the effect of the positive inside rule is stronger if positively charged residues are followed by a transmembrane helix. Such a mechanism obviously cannot aid C-terminal helices, and consequently, we find that the terminal helices in multi-spanning membrane proteins are more hydrophobic than internal helices.

An iron-regulated and glycosylation-dependent proteasomal degradation pathway for the plasma membrane metal transporter ZIP14.

Protein degradation is instrumental in regulating cellular function. Plasma membrane proteins targeted for degradation are internalized and sorted to multivesicular bodies, which fuse with lysosomes, where they are degraded. ZIP14 is a newly identified iron transporter with multitransmembrane domains. In an attempt to dissect the molecular mechanisms by which iron regulates ZIP14 levels, we found that ZIP14 is endocytosed, extracted from membranes, deglycosylated, and degraded by proteasomes. This pathway did not depend on the retrograde trafficking to the endoplasmic reticulum and thus did not involve the well-defined endoplasmic reticulum-associated protein degradation pathway. Iron inhibited membrane extraction of internalized ZIP14, resulting in higher steady-state levels of ZIP14. Asparagine-linked (N-linked) glycosylation of ZIP14, particularly the glycosylation at N102, was required for efficient membrane extraction of ZIP14 and therefore is necessary for its iron sensitivity. These findings highlight the importance of proteasomes in the degradation of endocytosed plasma membrane proteins.

In vitro synthesis of the E. coli Sec translocon from DNA.

Difficulties in constructing complex lipid/protein membranes have severely limited the development of functional artificial cells endowed with vital membrane-related functions. The Sec translocon membrane channel, which mediates the insertion of membrane proteins into the plasma membrane, was constructed in the membrane of lipid vesicles through in vitro expression of its component proteins. The components of the Sec translocon were synthesized from their respective genes in the presence of liposomes, thereby bringing about a functional complex. The synthesized E. coli Sec translocon mediated the membrane translocation of single- and multi-span membrane proteins. The successful translocation of a functional peptidase into the liposome lumen further confirmed the proper insertion of the translocon complex. Our results demonstrate the feasible construction of artificial cells, the membranes of which can be functionalized by directly decoding genetic information into membrane functions.

Immunization method for multi-pass membrane proteins using highly metastatic cell lines

A novel method using metastatic breast cancer cell lines was established for producing monoclonal antibodies (mAbs) against multi-span membrane proteins. Grafting of metastatic cells (MCF7-14) into the mammary gland of BALB/cJ/nu/nu mice induced splenic hypertrophy (1.6–3.0×108cells/spleen [n=6]). More than half of the mAbs against MCF7-14 cells reacted with the cell membrane. Inducing production of antibodies against the extracellular domain of multi-pass membrane proteins is difficult. Because the protein structure becomes more complex as the number of transmembrane domains increases, preparing antigens for immunization in which the original structure is maintained is challenging. Using highly metastatic MDA-MB231 cells as the host cell line, we produced mAbs against a 12 transmembrane protein, solute carrier family 6 member 6 (SLC6A6), as a model antigen. When SLC6A6-overexpressing MDA-MB231 cells were grafted into nude mice, the number of splenocytes increased to 2.7–11.4×108cells/spleen (n=10). Seven mAb-producing clones that not only recognized the extracellular domain of SLC6A6 but also were of the IgG subclass were obtained. Immunocytochemistry and flow cytometry analyses revealed that these mAbs recognized the native form of the extracellular domain of SLC6A6 on the cell surface. Our novel immunization method involving highly metastatic cells could be used to develop therapeutic mAbs against other multi-pass membrane proteins.