membrane orientation
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mSphere ◽  
2021 ◽  
Author(s):  
Paul-Adrian Bulzu ◽  
Vinicius Silva Kavagutti ◽  
Maria-Cecilia Chiriac ◽  
Charlotte D. Vavourakis ◽  
Keiichi Inoue ◽  
...  

Heliorhodopsins are enigmatic, novel rhodopsins with a membrane orientation that is opposite to all known rhodopsins. However, their cellular and ecological functions are unknown, and even their taxonomic distribution remains a subject of debate.


2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Benjamin A. Heitz ◽  
Robert Bränström ◽  
Wei Yang ◽  
Yiding Huang ◽  
Tilo Moede ◽  
...  

AbstractATP-sensitive K+ (KATP) channels couple cellular metabolism to electrical activity in many cell types. Wild-type KATP channels are comprised of four pore forming (Kir6.x) and four regulatory (sulfonylurea receptor, SURx) subunits that each contain RKR endoplasmic reticulum retention sequences that serve to properly translocate the channel to the plasma membrane. Truncated Kir6.x variants lacking RKR sequences facilitate plasma membrane expression of functional Kir6.x in the absence of SURx; however, the effects of channel truncation on plasma membrane orientation have not been explored. To investigate the role of truncation on plasma membrane orientation of ATP sensitive K+ channels, three truncated variants of Kir6.2 were used (Kir6.2ΔC26, 6xHis-Kir6.2ΔC26, and 6xHis-EGFP-Kir6.2ΔC26). Oocyte expression of Kir6.2ΔC26 shows the presence of a population of inverted inserted channels in the plasma membrane, which is not present when co-expressed with SUR1. Immunocytochemical staining of intact and permeabilized HEK293 cells revealed that the N-terminus of 6xHis-Kir6.2ΔC26 was accessible on both sides of the plasma membrane at roughly equivalent ratios, whereas the N-terminus of 6xHis-EGFP-Kir6.2Δ26 was only accessible on the intracellular face. In HEK293 cells, whole-cell electrophysiological recordings showed a ca. 50% reduction in K+ current upon addition of ATP to the extracellular solution for 6xHis-Kir6.2ΔC26, though sensitivity to extracellular ATP was not observed in 6xHis-EGFP-Kir6.2ΔC26. Importantly, the population of channels that is inverted exhibited similar function to properly inserted channels within the plasma membrane. Taken together, these data suggest that in the absence of SURx, inverted channels can be formed from truncated Kir6.x subunits that are functionally active which may provide a new model for testing pharmacological modulators of Kir6.x, but also indicates the need for added caution when using truncated Kir6.2 mutants.


2021 ◽  
Author(s):  
SIya Zhang ◽  
Hong-Yin Wang ◽  
Zhongwen Chen ◽  
Ilya Levental ◽  
Xubo Lin

Recently identified palmitoylation of PD-L1 is essential for immune regulation. To elucidate the underlying molecular mechanism, we performed giant plasma membrane vesicle (GPMV) experiments, us-scale all-atom molecular dynamics (MD) simulations and immune killing experiments. GPMV experiments indicated that PD-L1 palmitoylation enhanced its lipid raft affinity. MD simulations revealed dramatically different membrane orientation states of PD-L1 in liquid-ordered (L_o, lipid raft) compared to liquid-disordered (L_d, non-raft) membrane environments. L_d region promoted the lie-down orientation of PD-L1, which could inhibit its association with the PD-1 protein on immune cells and thus promote the immune killing of cancer cells. This hypothesis was supported by immune killing experiments using gamma-delta-T cells as effector cells and NCI-H1299 lung cancer cells as target cells. In short, our study demonstrates that the palmitoylation affects PD-L1 membrane localization and then membrane orientation, which thus regulates its binding with T cell PD-1 and the immune regulation. These observations may guide therapeutic strategies by explicating the regulation of immune checkpoint proteins by post-translational modifications and membrane environments.


2021 ◽  
Vol 75 (1) ◽  
Author(s):  
Andrey Rozenberg ◽  
Keiichi Inoue ◽  
Hideki Kandori ◽  
Oded Béjà

Microbial rhodopsins are diverse photoreceptive proteins containing a retinal chromophore and are found in all domains of cellular life and are even encoded in genomes of viruses. These rhodopsins make up two families: type 1 rhodopsins and the recently discovered heliorhodopsins. These families have seven transmembrane helices with similar structures but opposing membrane orientation. Microbial rhodopsins participate in a portfolio of light-driven energy and sensory transduction processes. In this review we present data collected over the last two decades about these rhodopsins and describe their diversity, functions, and biological and ecological roles. Expected final online publication date for the Annual Review of Microbiology, Volume 75 is October 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.


2020 ◽  
Vol 295 (48) ◽  
pp. 16370-16379
Author(s):  
Valerie Chen ◽  
Antonio E. Bruno ◽  
Laura L. Britt ◽  
Ciria C. Hernandez ◽  
Luis E. Gimenez ◽  
...  

The melanocortin receptor accessory protein 2 (MRAP2) plays a pivotal role in the regulation of several G protein–coupled receptors that are essential for energy balance and food intake. MRAP2 loss-of-function results in obesity in mammals. MRAP2 and its homolog MRAP1 have an unusual membrane topology and are the only known eukaryotic proteins that thread into the membrane in both orientations. In this study, we demonstrate that the conserved polybasic motif that dictates the membrane topology and dimerization of MRAP1 does not control the membrane orientation and dimerization of MRAP2. We also show that MRAP2 dimerizes through its transmembrane domain and can form higher-order oligomers that arrange MRAP2 monomers in a parallel orientation. Investigating the molecular details of MRAP2 structure is essential for understanding the mechanism by which it regulates G protein–coupled receptors and will aid in elucidating the pathways involved in metabolic dysfunction.


2020 ◽  
Author(s):  
Valerie Chen ◽  
Antonio E. Bruno ◽  
Laura L. Britt ◽  
Ciria C. Hernandez ◽  
Luis E. Gimenez ◽  
...  

ABSTRACTThe melanocortin receptor accessory protein 2 (MRAP2) plays a pivotal role in the regulation of several G-protein coupled receptors (GPCR) that are essential for energy balance and food intake. MRAP2 loss-of-function results in obesity in mammals. MRAP2 and its homolog MRAP1 have an unusual membrane topology and are the only known eukaryotic proteins that thread into the membrane in both orientations. In this study, we demonstrate that the conserved polybasic motif that dictates the membrane topology and dimerization of MRAP1 does not control the membrane orientation and dimerization of MRAP2. We also show that MRAP2 dimerizes through its transmembrane domain and can form higher order oligomers that arrange MRAP2 monomers in a parallel orientation. Investigating the molecular details of MRAP2 structure is essential for understanding the mechanism by which it regulates GPCRs and will aid in elucidating the pathways involved in metabolic dysfunction.


2020 ◽  
Vol 40 (5) ◽  
Author(s):  
Anja Nylander ◽  
Pawel Leznicki ◽  
Karina Vidovic ◽  
Stephen High ◽  
Martin L. Olsson

Abstract Antibodies to the Vel blood group antigen can cause adverse hemolytic reactions unless Vel-negative blood units are transfused. Since the genetic background of Vel-negativity was discovered in 2013, DNA-based typing of the 17-bp deletion causing the phenotype has facilitated identification of Vel-negative blood donors. SMIM1, the gene underlying Vel, encodes a 78-amino acid erythroid transmembrane protein of unknown function. The transmembrane orientation of SMIM1 has been debated since experimental data supported both the N- and C-termini being extracellular. Likewise, computational predictions of its orientation were divided and potential alternatives such as monotopic or dual-topology have been discussed but not investigated. We used a cell-free system to explore the topology of SMIM1 when synthesized in the endoplasmic reticulum (ER). SMIM1 was tagged with an opsin-derived N-glycosylation reporter at either the N- or C-terminus and synthesized in vitro using rabbit reticulocyte lysate supplemented with canine pancreatic microsomes as a source of ER membrane. SMIM1 topology was then determined by assessing the N-glycosylation of its N- or C-terminal tags. Complementary experiments were carried out by expressing the same SMIM1 variants in HEK293T/17 cells and establishing their membrane orientation by immunoblotting and flow cytometry. Our data consistently indicate that SMIM1 has its short C-terminus located extracellularly and that it most likely belongs to the tail-anchored class of membrane proteins with the bulk of the polypeptide located in the cytoplasm. Having established its membrane orientation in an independent model system, future work can now focus on functional aspects of SMIM1 as a potential regulator of erythropoiesis.


2019 ◽  
Vol 123 (41) ◽  
pp. 8644-8652 ◽  
Author(s):  
Priyanka Prakash ◽  
Alemayehu A. Gorfe

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