Transmembrane Region Of Protein Research Articles

Functional characterization of sugarcane ScFTIP1 reveals its role in Arabidopsis flowering

The timing of floral transition is essential for reproductive success in flowering plants. In sugarcane, flowering time affects the production of sugar and biomass. Although the function of the crucial floral pathway integrators, FLOWERING LOCUS T (FT), in sugarcane, has been uncovered, the proteins responsible for FT export and the underlying mechanism remain unexplored. In this study, we identified a member of the multiple C2 domain and transmembrane region proteins (MCTPs) family in sugarcane, FT-interacting protein 1 (ScFTIP1), which was localized to the endoplasmic reticulum. Ectopic expression of ScFTIP1 in the Arabidopsis mutant ftip1-1 rescued the late-flowering phenotype. ScFTIP1 interacted with AtFT in vitro and in vivo assays. Additionally, ScFTIP1 interacted with ScFT1 and the floral inducer ScFT3. Furthermore, we found that the NAC member, ScNAC23, could directly bind to the ScFTIP1 promoter and negatively regulate its transcription. Overall, our findings revealed the function of ScFTIP1 and proposed a potential mechanism underlying flowering regulation in sugarcane.

Precise assembly of inside-out cell membrane camouflaged nanoparticles via bioorthogonal reactions for improving drug leads capturing.

Cell membrane camouflaged nanoparticles have been widely used in the field of drug leads discovery attribute to their unique biointerface targeting function. However, random orientation of cell membrane coating does not guarantee effective and appropriate binding of drugs to specific sites, especially when applied to intracellular regions of transmembrane proteins. Bioorthogonal reactions have been rapidly developed as a specific and reliable method for cell membrane functionalization without disturbing living biosystem. Herein, inside-out cell membrane camouflaged magnetic nanoparticles (IOCMMNPs) were accurately constructed via bioorthogonal reactions to screen small molecule inhibitors targeting intracellular tyrosine kinase domain of vascular endothelial growth factor recptor-2. Azide functionalized cell membrane acted as a platform for specific covalently coupling with alkynyl functionalized magnetic Fe3O4 nanoparticles to prepare IOCMMNPs. The inside-out orientation of cell membrane was successfully verified by immunogold staining and sialic acid quantification assay. Ultimately, two compounds, senkyunolide A and ligustilidel, were successfully captured, and their potential antiproliferative activities were further testified by pharmacological experiments. It is anticipated that the proposed inside-out cell membrane coating strategy endows tremendous versatility for engineering cell membrane camouflaged nanoparticles and promotes the development of drug leads discovery platforms.

A Post-translational Modification-enhanced Pull-down Method to Study Degron Domains and the Associated Protein Degradation Complexes.

The identification and characterization of the ubiquitin E-ligase complexes involved in specific proteins' degradation via the ubiquitin-proteasome system (UPS) can be challenging and require biochemical purification processes and in vitro reconstitution assays. Likewise, evaluating the effect of parallel phosphorylation and ubiquitination events occurring in vivo at dual phospho/ubiquitin-regulated motifs (called Phospho-Degrons or pDegrons) driving UPS degradation of the targeted protein has remained elusive. Indeed, the functional study of such E1-E2-E3 complexes acting on a protein-specific level requires previously or otherwise acquired knowledge of the nature of such degradation complex components. Furthermore, the molecular basis of the interaction between an E3 ligase and its pDegron binding motif on a target protein would require individually optimized in vitro kinase and ubiquitination assays. Here, we describe a novel enzymatically enhanced pull-down method to functionally streamline the discovery and functional validation of the ubiquitin E-ligase components interacting with a phospho-degron containing protein domain and/or sub-domain. The protocol combines key features of a protein kinase and ubiquitination in vitro assay by including them in a pull-down step exerted by a known or putative pDegron-tagged peptide using the cell extracts as a source of enzymatically active post-translational modification (PTM) modifying/binding native proteins. The same method allows studying specific stimuli or treatments towards the recruitment of the molecular degradation complex at the target protein's phospho-degron site, reflecting in vivo-initiated events further enhanced through the assay design. In order to take full advantage of the method over traditional protein-protein interaction methods, we propose to use this PTM-enhanced (PTMe) pull down both towards the degradation complex discovery/ID phase as well as for the functional pDegron recruitment validation phase, which is the one described in the present protocol both graphically and in a stepwise fashion for reproduceable results. Key features • Suitable to study UPS-regulated (a) cytosolic and/or nuclear proteins, (b) intracellular region of transmembrane proteins, and (c) protein sub-domains bearing a known/putative pDegron motif. • Requires a biotin-tagged recombinant version of the target protein and/or sub-domain. • Allows the qualitative and quantitative analysis of endogenous ubiquitin (Ub) E-ligases recruitment to a known or putative pDegron bearing protein/sub-domain. • Allows simultaneous testing of various treatments and/or conditions affecting the phosphorylative and/or ubiquitylation status of the studied pDegron bearing protein/sub-domain and the recruited factors. Graphical overview.

Immunoinformatic analysis of the whole proteome for vaccine design: An application to Clostridium perfringens.

Clostridium perfringens is a dangerous bacterium and known biological warfare weapon associated with several diseases, whose lethal toxins can produce necrosis in humans. However, there is no safe and fully effective vaccine against C. perfringens for humans yet. To address this problem, we computationally screened its whole proteome, identifying highly immunogenic proteins, domains, and epitopes. First, we identified that the proteins with the highest epitope density are Collagenase A, Exo-alpha-sialidase, alpha n-acetylglucosaminidase and hyaluronoglucosaminidase, representing potential recombinant vaccine candidates. Second, we further explored the toxins, finding that the non-toxic domain of Perfringolysin O is enriched in CTL and HTL epitopes. This domain could be used as a potential sub-unit vaccine to combat gas gangrene. And third, we designed a multi-epitope protein containing 24 HTL-epitopes and 34 CTL-epitopes from extracellular regions of transmembrane proteins. Also, we analyzed the structural properties of this novel protein using molecular dynamics. Altogether, we are presenting a thorough immunoinformatic exploration of the whole proteome of C. perfringens, as well as promising whole-protein, domain-based and multi-epitope vaccine candidates. These can be evaluated in preclinical trials to assess their immunogenicity and protection against C. perfringens infection.

Characterization and phylogenetic analysis of multiple C2 domain and transmembrane region proteins in maize

BackgroundMultiple C2 domain and transmembrane region proteins (MCTPs) are evolutionarily conserved and important signaling molecules. However, the MCTP gene family has not been comprehensively analyzed in maize.ResultsIn this study, 385 MCTP genes were identified in all surveyed 38 species. Moreover, gene duplication mode exploration showed that whole genome duplication (WGD) mainly contributed to the expansion of MCTP genes in angiosperms. Phylogeny reconstruction with all surveyed species by the maximum-likelihood (ML) method showed five clades of MCTPs, Clades I to V. Each clade of MCTPs had conservative structures and motifs. Focusing on maize, 17 MCTPs were identified, and a neighborjoining (NJ) phylogenetic tree with only ZmMCTPs was also constructed. As expected, 17 MCTPs showed similar phylogenetic relationships in the neighbor-joining (NJ) tree with those in the maximum-likelihood (ML) tree and could also be divided into five subclades. Moreover, ZmMCTP members in different clades showed specific gene structure, conserved motif, and domain structure compositions. Intriguingly, most ZmMCTP genes were intronless. Analyses of isoelectric points (pIs) and grand averages of hydropathicity (GRAVYs) indicated that the N-terminus was more dispersive than the C-terminus. Further tissue-specific expression analysis indicated that duplicated ZmMCTP pairs involved in whole genome duplication (WGD) had similar expression trends. Finally, ZmMCTPs were transcriptionally altered under diverse abiotic stresses and hormone treatments.ConclusionsOur results contribute to deciphering the evolutionary history of MCTPs in maize and other plants, facilitating further functional analysis of these factors, and provide a basis for further clarification of the molecular mechanism of stress responses.

Analysis of the MCTP Amino Acid Sequence Reveals the Conservation of Putative Calcium- and Lipid-Binding Pockets Within the C2 Domains In Silico.

MCTPs (Multiple C2 Domains and Transmembrane region Proteins) are evolutionarily and structurally related to other C2 proteins, which are central to exocytosis and membrane trafficking; however, their specific function has been little studied. MCTPs are associated with endosomes and the endoplasmic reticulum and possess three C2 domains (C2A-C2C) and two transmembrane regions (TMRs) well conserved in different species. Here, we generated structural models of the MCTP C2 domains of C. elegans and analyzed their putative function by docking, which revealed that these domains possess Ca2+- and lipid-binding pockets, suggesting that MCTPs play a significant, calcium-dependent role in membrane physiology.

Global Identification and Characterization of C2 Domain-Containing Proteins Associated with Abiotic Stress Response in Rice (Oryza sativa L.).

C2 domain-containing proteins (C2DPs) have been identified in different genomes that contain single or multiple C2 domains in their C- or N-terminal. It possesses higher functional activity in the transmembrane regions. The identification of C2 domains were reported in a previous study, such as multiple C2 domains and transmembrane-region proteins (MCTPs) and N-terminal-TM-C2 domain proteins (NTMC2s) of rice, Arabidopsis thaliana, and cotton, whereas the C2DP gene family in rice has not been comprehensively studied, and the role of the C2DP gene in rice in response to abiotic stress is not yet fully understood. In this study, we identified 82 C2DPs in the rice genome and divided them into seven groups through phylogenetic analysis. The synteny analysis revealed that duplication events were either exhibited within the genome of rice or between the genomes of rice and other species. Through the analysis of cis-acting elements in promoters, expression profiles, and qRT-PCR results, the functions of OsC2DPs were found to be widely distributed in diverse tissues and were extensively involved in phytohormones-related and abiotic stresses response in rice. The prediction of the microRNA (miRNA) targets of OsC2DPs revealed the possibility of regulation by consistent miRNAs. Notably, OsC2DP50/51/52 as a co-tandem duplication exhibited similar expression variations and involved the coincident miRNA-regulation pathway. Moreover, the results of the genotypic variation and haplotype analysis revealed that OsC2DP17, OsC2DP29, and OsC2DP49 were associated with cold stress responses. These findings provided comprehensive insights for characterizations of OsC2DPs in rice as well as for their roles for abiotic stress.

Family-Wide Evaluation of Multiple C2 Domain and Transmembrane Region Protein in Gossypium hirsutum.

Multiple C2 domain and transmembrane region proteins (MCTPs) are a group of evolutionarily conserved proteins and show emerging roles in mediating protein trafficking and signaling transduction. Although, several studies showed that MCTPs play important roles during plant growth and development, their biological functions in cotton remain largely unknown. Here, we identify and characterize 33 GhMCTP genes from upland cotton (Gossypium hirsutum) and reveal the diverse expression patterns of GhMCTPs in various tissues. We also find that GhMCTP7, GhMCTP12, and GhMCTP17 are highly expressed in the main stem apex, suggesting their possible roles in shoot development. Through analyzing different cotton species, we discover plant heights are closely related to the expression levels of GhMCTP7, GhMCTP12, and GhMCTP17. Furthermore, we silence the expression of GhMCTP genes using virus-induced gene silencing (VIGS) system in cotton and find that GhMCTP7, GhMCTP12, and GhMCTP17 play an essential role in shoot meristem development. GhMCTPs interact with GhKNAT1 and GhKNAT2 and regulate meristem development through integrating multiple signal pathways. Taken together, our results demonstrate functional redundancy of GhMCTPs in cotton shoot meristem development and provide a valuable resource to further study various functions of GhMCTPs in plant growth and development.

Saturation Mutagenesis of the Transmembrane Region of HokC in Escherichia coli Reveals Its High Tolerance to Mutations.

Cells adapt to different stress conditions, such as the antibiotics presence. This adaptation sometimes is achieved by changing relevant protein positions, of which the mutability is limited by structural constrains. Understanding the basis of these constrains represent an important challenge for both basic science and potential biotechnological applications. To study these constraints, we performed a systematic saturation mutagenesis of the transmembrane region of HokC, a toxin used by Escherichia coli to control its own population, and observed that 92% of single-point mutations are tolerated and that all the non-tolerated mutations have compensatory mutations that reverse their effect. We provide experimental evidence that HokC accumulates multiple compensatory mutations that are found as correlated mutations in the HokC family multiple sequence alignment. In agreement with these observations, transmembrane proteins show higher probability to present correlated mutations and are less densely packed locally than globular proteins; previous mutagenesis results on transmembrane proteins further support our observations on the high tolerability to mutations of transmembrane regions of proteins. Thus, our experimental results reveal the HokC transmembrane region high tolerance to loss-of-function mutations that is associated with low sequence conservation and high rate of correlated mutations in the HokC family sequences alignment, which are features shared with other transmembrane proteins.

Organelle homeostasis principles: How organelle quality control and inter-organelle crosstalk promote cell survival.

To survive, cells sense their surroundings and adapt to enable homeostasis. Studies dissecting this process reveal organizational principles, including quality-control pathways, changes to organelle shape, and inter-organelle communication, that facilitate metabolic or developmental remodeling. In this issue, several reviews discuss these organelle homeostasis principles and how they are altered in disease.

Florigen trafficking integrates photoperiod and temperature signals in Arabidopsis.

The transition to flowering is the most dramatic phase change in flowering plants and is crucial for reproductive success. A complex regulatory network in plants has evolved to perceive and integrate the endogenous and environmental signals. These signals perceived, including day length and temperature, converge to regulate FLOWERING LOCUS T (FT), which encodes a mobile stimulus required for floral induction in Arabidopsis. Despite the discovery of modulation of FT messenger RNA (mRNA) expression by ambient temperature, whether the trafficking of FT protein is controlled in response to changes in growth temperature is so far unknown. Here, we show that FT transport from companion cells to sieve elements is controlled in a temperature-dependent manner. This process is mediated by multiple C2 domain and transmembrane region proteins (MCTPs) and a soluble N-ethylmaleimide-sensitive factor protein attachment protein receptor (SNARE). Our findings suggest that ambient temperatures regulate both FT mRNA expression and FT protein trafficking to prevent precocious flowering at low temperatures and ensure plant reproductive success under favorable environmental conditions.

Genome-wide identification and characterization of multiple C2 domains and transmembrane region proteins in Gossypium hirsutum

BackgroundMultiple C2 domains and transmembrane region proteins (MCTPs) may act as transport mediators of other regulators. Although increased number of MCTPs in higher plants implies their diverse and specific functions in plant growth and development, only a few plant MCTPs have been studied and no study on the MCTPs in cotton has been reported.ResultsIn this study, we identified 31 MCTPs in G. hirsutum, which were classified into five subfamilies according to the phylogenetic analysis. GhMCTPs from subfamily V exhibited isoelectric points (pIs) less than 7, whereas GhMCTPs from subfamily I, II, III and IV exhibited pIs more than 7.5, implying their distinct biological functions. In addition, GhMCTPs within subfamily III, IV and V exhibited more diverse physicochemical properties, domain architectures and expression patterns than GhMCTPs within subfamily I and II, suggesting that GhMCTPs within subfamily III, IV and V diverged to perform more diverse and specific functions. Analyses of conserved motifs and pIs indicated that the N-terminus was more divergent than the C-terminus and GhMCTPs’ functional divergence might be mainly contributed by the N-terminus. Furthermore, yeast two-hybrid assay indicated that the N-terminus was responsible to interact with target proteins. Phylogenetic analysis classified multiple N-terminal C2 domains into four subclades, suggesting that these C2 domains performed different molecular functions in mediating the transport of target proteins.ConclusionsOur systematic characterization of MCTPs in G. hirsutum will provide helpful information to further research GhMCTPs’ molecular roles in mediating other regulators’ transport to coordinate growth and development of various cotton tissues.

TraPS-VarI: Identifying genetic variants altering phosphotyrosine based signalling motifs

Patient stratification and individualized therapeutic strategies rely on the established knowledge of genotype-specific molecular and cellular alterations of biological and therapeutic significance. Whilst almost all approved drugs have been developed based on the Reference Sequence protein database (RefSeq), the latest genome sequencing studies establish the substantial prevalence of non-synonymous genetic mutations in the general population, including stop-insertion and frame shift mutations within the coding regions of membrane proteins. While the availability of individual genotypes are becoming increasingly common, the biological and clinical interpretations of mutations among individual genomes is largely lagging behind. Lately, transmembrane proteins of haematopoietic (myeloid and lymphoid) derived immune cells have attracted much attention as important targets for cancer immunotherapies. As such, the signalling properties of haematological transmembrane receptors rely on the membrane-proximal phosphotyrosine based sequence motifs (TBSMs) such as ITAM (immunoreceptor tyrosine-based activation motif), ITIM (immunoreceptor tyrosine-based inhibition motif) and signal transducer and activator of transcription 3 (STAT3)-recruiting YxxQ motifs. However, mutations that alter the coding regions of transmembrane proteins, resulting in either insertion or deletion of crucial signal modulating TBSMs, remains unknown. To conveniently identify individual cell line-specific or patient-specific membrane protein altering mutations, we present the Transmembrane Protein Sequence Variant Identifier (TraPS-VarI). TraPS-VarI is an annotation tool for accurate mapping of the effect of an individual’s mutation in the transmembrane protein sequence, and to identify the prevalence of TBSMs. TraPS-VarI is a biologist and clinician-friendly algorithm with a web interface and an associated database browser (https://traps-vari.readthedocs.io/).

Genome-wide identification and phylogenetic analysis of rice FTIP gene family

FT-INTERACTING PROTEIN (FTIP) gene family in rice are the members of multiple C2 domain and transmembrane region proteins (MCTPs). There are many homologs of OsFTIPs in plants; however, the bioinformatics of them remains unclear. In the studies, 13 OsFTIP genes are identified in rice. OsFTIPs are unevenly located in 12 chromosomes. The OsFTIPs are phylogenetically divided into three clades. Cis-elements respond to abiotic stress, light, and hormones are found in the promoter region of OsFTIPs which are induced by the stimuli. All OsFTIPs are expressed with different profiles. Syntenic analysis of 128 OsFTIPs and FTIP-like homologs reveals that various number of gene pairs are identified between rice and other species. The 128 FTIP-like homologs are divided into six groups which fall into three classes. Ten motifs are shared by most OsFTIPs and their homologs. The studies provide a theoretical basis for further elucidating the functions of OsFTIP gene family.

The MCTP-SNARE Complex Regulates Florigen Transport in Arabidopsis.

Multiple flowering pathways in Arabidopsis (Arabidopsis thaliana) converge on the transcriptional regulation of FLOWERING LOCUS T (FT), encoding a mobile floral stimulus that moves from leaves to the shoot apex. Despite our progress in understanding FT movement, the mechanisms underlying its transport along the endoplasmic reticulum-plasmalemma pathway in phloem companion cells remain largely unclear. Here, we show that the plasma membrane-resident syntaxin-like glutamine-soluble N-ethylmaleimide-sensitive factor protein attachment protein receptor (Q-SNARE), SYNTAXIN OF PLANTS121 (SYP121), interacts with QUIRKY (QKY), a member of the family of multiple C2 domain and transmembrane region proteins (MCTPs), to mediate FT transport in Arabidopsis. QKY and SYP121 coordinately regulate FT movement to the plasmalemma through the endosomal trafficking pathway and are required for FT export from companion cells to sieve elements, thus affecting FT transport through the phloem to the shoot apical meristem. These findings suggest that MCTP-SNARE complex-mediated endosomal trafficking is essential for the export of florigen from phloem companion cells to sieve elements to induce flowering.plantcell;31/10/2475/FX1F1fx1.

Prediction of disease-associated mutations in the transmembrane regions of proteins with known 3D structure.

Being able to assess the phenotypic effects of mutations is a much required capability in precision medicine. However, most of the currently available structure-based methods actually predict stability changes caused by mutations rather than their pathogenic potential. There are also no dedicated methods to predict damaging mutations specifically in transmembrane proteins. In this study we developed and applied a machine-learning approach to discriminate between disease-associated and benign point mutations in the transmembrane regions of proteins with known 3D structure. The method, called BorodaTM (BOosted RegressiOn trees for Disease-Associated mutations in TransMembrane proteins), was trained on sequence-, structure-, and energy-derived descriptors. When compared with the state-of-the-art methods, BorodaTM is superior in classifying point mutations in transmembrane regions. Using BorodaTM we have conducted a large-scale mutation analysis in the transmembrane regions of human proteins with known 3D structures. For each protein we generated structural models for all point mutations by replacing each residue to 19 possible residue types. We classified ~1.8 millions point mutations as benign or diseased-associated and made all predictions available as a Web-server at https://www.iitm.ac.in/bioinfo/MutHTP/boroda.php.

Benchmarking subcellular localization and variant tolerance predictors on membrane proteins

BackgroundMembrane proteins constitute up to 30% of the human proteome. These proteins have special properties because the transmembrane segments are embedded into lipid bilayer while extramembranous parts are in different environments. Membrane proteins have several functions and are involved in numerous diseases. A large number of prediction methods have been introduced to predict protein subcellular localization as well as the tolerance or pathogenicity of amino acid substitutions.ResultsWe tested the performance of 22 tolerance predictors by collecting information on membrane proteins and variants in them. The analysis indicated that the best tools had similar prediction performance on transmembrane, inside and outside regions of transmembrane proteins and comparable to overall prediction performances for all types of proteins. PON-P2 had the highest performance followed by REVEL, MetaSVM and VEST3. Further, we tested with the high quality dataset also the performance of seven subcellular localization predictors on membrane proteins. We assessed separately the performance for single pass and multi pass membrane proteins. Predictions for multi pass proteins were more reliable than those for single pass proteins.ConclusionsThe predictors for variant effects had better performance than subcellular localization tools. The best tolerance predictors are highly reliable. As there are large differences in the performances of tools, end-users have to be cautious in method selection.

In silico analysis of transmembrane protein 31 (TMEM31) antigen to design novel multiepitope peptide and DNA cancer vaccines against melanoma

Multiepitope cancer vaccines are announcing themselves as the future of melanoma treatment. Herein, high immunogenic regions of transmembrane protein 31 (TMEM31) antigen were selected according to cytotoxic T lymphocytes’ (CTL) epitopes and major histocompatibility complex (MHC) binding affinity through in silico analyses. The 32–62, 77–105, and 125–165 residues of the TMEM31 were selected as the immunodominant fragments. They were linked together by RVRR and HEYGAEALERAG motifs to improve epitopes separation and presentation. In addition, to activate helper T lymphocytes (HTL), Pan HLA DR-binding epitope (PADRE) peptide sequence and tetanus toxin fragment C (TTFrC) were incorporated into the final construct. Also, the Beta-defensin conserved domain was utilized in the final construct as a novel adjuvant for Toll-like receptor 4/myeloid differentiation factor (TLR4-MD) activation. The CTL epitopes, cleavage sites, post-translational modifications, TAP transport efficiency, and B cells epitopes were predicted for the peptide vaccine. The final construct contained multiple CTL and B cell epitopes. In addition, it showed 93.55% and 99.13% population coverage in the world for HLA I and HLA II, respectively. According to these preliminary results, the multiepitope cancer vaccine can be an appropriate choice for further experimental investigations.

Transmembrane protein 64 modulates prostate tumor progression by regulating Wnt3a secretion.

Wnt3a is a glycosylated ligand that activates the β-catenin-dependent signaling pathway. Wnt signaling is also important in the prostate tumor microenvironment, and Wnt proteins secreted by the tumor stroma promote resistance to therapy. Bioactive Wnt3a production requires a number of dedicated factors in the secretory cell, but their coordinated functions are not fully understood. We previously reported transmembrane protein 64 (Tmem64) as a novel regulator of the Wnt/β-catenin signaling pathway, which is correlated with β-catenin regulation. In the present study, the role of Tmem64 in prostate cancer cells was investigated by modulating Wnt3a secretion. Overexpression of Tmem64 inhibited Wnt3a secretion and Lef/Tcf-sensitive transcription. By contrast, a Tmem64 mutation deleting the protein's transmembrane region restored Wnt3a secretion. Notably, Tmem64 protein and mRNA in PC3 cells were significantly overexpressed compared with that observed in LNCaP and DU145 cells. In a mouse metastasis model intracardially injected with PC3 cells, Tmem64 expression was downregulated in the metastatic spine and mandible lesions compared with in the primary injection regions. However, Wnt3a was strongly expressed in the metastatic spine and mandible lesions. Collectively, these findings suggest that Tmem64 is involved in the metastatic progression of prostate cancer cells by regulating Wnt3a secretion.

Vesicular stomatitis virus G protein transmembrane region is crucial for the hemi-fusion to full fusion transition

Viral fusion proteins are essential for enveloped virus infection. These proteins mediate fusion between the virus envelope and host cellular membrane to release the viral genome into cells. Vesicular stomatitis virus G (VSV G) protein is a typical type III viral fusion protein. To study the mechanism of VSV G protein mediated membrane fusion, we set up a cell-cell fusion system in which cells are marked by different fluorescent proteins. Taking advantage of this system, we performed real-time recording and quantitative analysis of the cell fusion mediated by VSV G. We found that the time scale required for VSV G mediated cell-cell fusion was approximately 1–2 minutes. Next, we specifically examined the function of the transmembrane (TM) region of VSV G protein in membrane fusion by replacing the TM region with those of other fusion proteins. The TM region replacements dramatically impaired VSV G protein function in the cell-cell fusion assay and diminished VSV G mediated lentivirus and recombinant VSV infection efficiency. Further experiments implied that the TM region played a role in the transition from hemi-fusion to full fusion. Several residues within the TM region were identified as important for membrane fusion. Overall, our findings unraveled the important function of the TM region in VSV G mediated viral fusion.