Single Helix Research Articles

From Double-Stranded Helicates to Abiotic Double Helical Supramolecular Assemblies.

The folding of oligomeric strands is the method that nature has selected to generate ordered assemblies presenting spectacular functions. In the purpose to mimic these biomacro-molecules and extend their properties and functions, chemists make important efforts to prepare artificial secondary, tertiary, and even quarternary structures based on folded abiotic backbones. A large variety of oligomers and polymers, encoded with chemical informations, were designed, synthesized and characterized, and the establishment of non-covalent interactions lead to complex and functional supramolecular architectures resulting from a spontaneous self-assembly process. The association of comple-mentary molecular strands into double helical structures is a common structural pattern of nucleic acids and proteins, so the synthesis of bio-inspired double helices has emerged as an important subject. In recent years, a number of synthetic oligo-mers have been reported to form stable double helices and it was shown that the equilibrium between single and double helices can be controlled via different stimuli like the modification of the solvent or the temperature. This kind of structure presents highly interesting functions, such as molecular recognition within the cavity of double helices, and some other potential applications will emerge in the future.

Evolution of Peptidomimetics-Based Chiral Assemblies of β-Sheet, α-Helix, and Double Helix Involving Chalcogen Bonds.

Developing chiral assemblies that mimic biological secondary structures, e.g., protein β-sheet, α-helix, and DNA double helix, is a captivating goal in supramolecular chemistry. Here, we create a family of biomimetic chiral assemblies from alanine-based peptidomimetics, wherein the incorporation of N-terminal 2,1,3-benzoselenadiazole groups enables the rarely utilized chalcogen bonding as the adhesive interaction. While the alanine-based acylhydrazine molecule 1L was designed as a building unit with an extended conformation, simple derivatization of 1L affords folded unilateral N-amidothiourea 2L with one β-turn and bilateral N-amidothiourea 3L with two β-turns. This derivatization leads to the evolution of molecular assemblies from β-sheet organization (1L) to single helix (α-helix mimic, 2L) and ultimately to double helix (3L), illustrating an evolutionary route relating the structures and superstructures. In the case of the double helix formed by 3L, an unexpected cis-form that brings the two β-turns into one side was observed, stabilized via the π···π interaction between two N-terminal 2,1,3-benzoselenadiazole groups. This conformation allows double-crossed N-Se···S═C chalcogen bonds to support a DNA-like P-double helix featuring intrastrand noncovalent interactions and interstrand covalent linkages, surviving in both the solid state and in dilute acetonitrile solution phase.

Biophysical analysis of the membrane-proximal Venus Flytrap domain of ESAG4 receptor-like adenylate cyclase from Trypanosoma brucei

The protozoan parasite Trypanosoma brucei possesses a large family of transmembrane receptor-like adenylate cyclases (RACs), primarily located to the flagellar surface and involved in sensing of the extracellular environment. RACs exhibit a conserved topology characterized by a large N-terminal extracellular moiety harbouring two Venus Flytrap (VFT) bilobate structures separated from an intracellular catalytic domain by a single transmembrane helix. RAC activation, which typically occurs under mild acid stress, requires the dimerization of the intracellular catalytic domain. The occurrence of VFT domains in the RAC’s extracellular moiety suggests their potential responsiveness to extracellular ligands in the absence of stress, although no such ligands have been identified so far. Herein we report the biophysical characterization of the membrane-proximal VFT2 domain of a bloodstream form-specific RAC called ESAG4, whose ectodomain 3D structure is completely unknown. The paper describes an AlphaFold2-based optimisation of the expression construct, enabling facile and high-yield recombinant production and purification of the target protein. Through an interdisciplinary approach combining various biophysical methods, we demonstrate that the optimised VFT2 domain obtained by recombination is properly folded and behaves as a monomer in solution. The latter suggests a ligand-binding capacity independent of dimerization, unlike typical mammalian VFT receptors, as guanylate cyclase. In silico VFT2 genomic analyses shows divergence among cyclase isoforms, hinting at ligand specificity. Taken together this improved procedure enabling facile and high-yield recombinant production and purification of the target protein could benefit researchers studying trypanosomal RAC VFT domains but also any trypanosome domain with poorly defined boundaries. Additionally, our findings support the stable monomeric VFT2 domain as a useful tool for future structural investigations and ligand screening.

Mechanism underlying V-type structure formation in maize starch through glycerol–ethanol thermal substitution method

V-type starches have been widely used in food science, agriculture, and biomedical science, but their mechanism of formation in nonaqueous solvents remains unclear. This study performed glycerol–ethanol thermal substitution to prepare V-type starch at atmospheric pressure. Scanning electron microscopy and confocal laser scanning microscopy revealed that breakage of starch particles began in the hilum region and rapidly spread to the periphery with the temperature increased from 100 °C to 130 °C. The Maltese crosses of the starch disappeared and starch particles had the form of doughnut-shaped rings when the glycerol temperature was 140 °C. Glycerol temperature was not found to significantly affect the chain length of amylopectin, meaning that glycerol molecules may stabilize the conformation of amylose through hydrogen bonding, promoting the amylose to form a V-type spiral structure. Additionally, A-type crystallinity and double helix structure proportion of the samples significantly decreased from 30.81 % and 42.21 % to 5.70 % and 16.47 % as the temperature of glycerol was increased to 140 °C, whereas the V-type crystallinity and single helix structure proportion of the samples remarkably increased from 13.26 % and 1.05 % to 37.97 % and 25.09 %, respectively. This study provided a facile and versatile approach to effectively regulate the formation of V-type starch.

Synthesis, structures, and photoluminescence of zinc(II) coordination polymers based on carboxylates and the semirigid ligand 1,4-bis(2-methylbenzimidazol-1-ylmethyl)benzene

Two fluorescent Zn(II) polymers (1 and 2) with different dicarboxylic acid co-ligands have been obtained under hydrothermal reaction conditions. In 1 and 2, the flexible bmb adopts two trans-conformations with different Ndonor···N–Csp3···Csp3 torsion angles, generating 3-D interspersed (412·63)-pcu grids. The dicarboxylic acid co-ligands influence the conformation of bmb, which lead to diverse spatial structures from a single helix to a 2-D lattice network. Photoluminescence investigations reveal that 1 has a redshift compared with o-H2oba (4,4'-oxydibenzoic acid) and 2 has a blueshift of 20 nm compared with H2bpdc ([1,1'-biphenyl]-4,4'-dicarboxylic acid), which can be attributed to a ligand-centered charge transfer.

ARCIMBOLDO at low resolution: Verification for coiled coils and globular proteins.

Crystallography at low resolution must determine the atomic model from less experimental observations, which is challenging in the absence of a model. In addition, model bias is more severe when independent experimental data are scarce. Our methods solve the phase problem by combining the location of accurate model fragments using Phaser with density modification and interpretation of the resulting maps using SHELXE. From a partial, correct structure, the density modification process and the stereochemical constraints draw the rest of the structure, validating the result. This same principle is now exploited at low resolution. Coiled coils are important, ubiquitous structures but notoriously difficult to phase and to predict. Both correct solutions and incorrect ones are poorly discriminated by the crystallographic figures of merit as long as helices are correctly oriented. We incorporate coiled-coil verification, designed to set up competing, incompatible structural hypotheses to probe both the results and establish the power of the data to discriminate them. Efficiency of coiled-coil phasing and validation in test cases from 3 to 4 Å is demonstrated in ARCIMBOLDO_LITE, placing single helices, and in ARCIMBOLDO_SHREDDER, with fragments derived from AlphaFold models. SHELXE tracing at low resolution has been enhanced, maintaining its local character but extending the environment assessment. For non-helical structures, verification is demonstrated in the fragment location process. Its use is exemplified with the solution of the VSR1 structure at 3.5 Å, depending on LLG optimization and the emergence of new features in the electron density. Relying on verification, we have extended the use of the ARCIMBOLDO software to low resolution.

Potassium-induced κ-carrageenan helices resist degradation by gut microbiota in an in vitro model

Higher-order structures hierarchy of κ-carrageenan in the presence of potassium was reported. Yet, the destiny of κ-carrageenan in higher-order structures in the colon and their impacts on the gut microbiota community remains to be revealed. This study aims to investigate the fermentation behaviors and trace the structural change of higher-order structures of κ-carrageenan during in vitro fermentation. Herein, κ-carrageenan gels were induced in the presence of 10, 40, and 70 mmol/L potassium ions, and a structural-hierarchical transition from secondary to quaternary structures was observed using an AFM and TEM. Results from GPC-MALLS, TLC, IEC, and GC indicated that Single helices were partially fermented by fecal microbiota, while supercoiled helices (tertiary structures) and intertwined helices networks (quaternary structures) were hardly fermented. Tracing the structural change of higher-order κ-carrageenan suggested that the single helices were partially deformed, as shown by the 60% reduction of single helices width, while supercoiled helices were more resistant to fermentation compared to single helices. The super-strands networks were hardly fermented as their width distribution center had been sustained at around 90 nm from 12 to 48 h. In addition, the 16S rRNA gene (V3-V4) amplicon sequencing results indicated that Bifidobacterium longum showed a positive correlation to the structural hierarchy (R = 0.734, p < 0.01). Together, the higher-order structures hierarchy of κ-carrageenan can resist degradation by human gut microbiota.

Molecular motion behaviors of starch affect starch-polyphenol inclusion complex and digestibility among different stilbenes polyphenol structures

Starch-polyphenol V-type inclusion complex has become a hot topic due to its anti-digestibility and nutritional function. This paper aimed to explore the molecular motion behavior of starch affects starch-polyphenol inclusion complex and digestibility among different stilbene polyphenol structures (resveratrol (RA), pterostilbene (PB) and polydatin (PD) via the high-pressure homogenization (HPH) and heat moisture treatment (HMT) processes), which represented the fully extended and limited molecular motion behavior of starch, respectively. These results revealed distinct trends in complex formation among different stilbenes polyphenol structures, highlighting RA as particularly conducive to increasing single helix and V-type crystalline structures with the highest resistant starch (RS) content of 28.11 % due to its smaller steric hindrance. Novelty, in HPH environments with extended molecular motion behavior, the steric hindrance and hydrophobicity/CH-π interactions of polyphenols influence complex formation in the order of RA > PB > PD. Conversely, in HMT systems with limited molecular motion behavior, the limited movement of molecules emphasized the importance of hydrogen bond interactions between polyphenols and starch. Thus, the glucoside in PD enhanced its interaction with starch compared to methoxy-modified PB, leading to increased formation of inclusion complex with RS content of 18.61 %. Overall, these findings deepen the understanding of starch-polyphenol interactions.

Upcycling of industrial pea starch by rapid spray nanoprecipitation to develop plant-derived oil encapsulated starch nanoparticles for potential agricultural applications

Starch is one of the natural encapsulant materials widely used in food, pharmaceutical and cosmetic industries. Starch with high amylose content (above 40 %, w/w) is prone to form single helices V-type allomorph with a hydrophilic outer surface and a hydrophobic inner cavity making them suitable for encapsulation of hydrophobic compounds such as essential oils, fatty acids, and vitamins. Pea starch obtained from pea protein processing industries have a high amylose content (40 %, w/w) rendering them unsuitable for direct food applications as ingredients. Therefore, in this study, an in-house spraying procedure was used to synthesize nanoparticles using pea starch, to encapsulate neem oil, a natural antimicrobial compound obtained from neem plant (Azadirachta indica) seed. The synthesis of the oil-encapsulated starch nanoparticles (OESNP) was optimized using a Box-Behnken experimental design to study the influence of the processing parameters such as the initial starch concentration, homogenization speed, duration of homogenization, sample injection rate, and quantity of antisolvent (ethanol). The optimized sample showed an 80–90 % encapsulation efficiency and particle size of <500 nm. The spherical OESNPs also demonstrated sustained release of the oil compared to free oil when dispersed in water. X-ray diffraction analysis revealed the coexistence of C-type and V-type polymorphs in the loaded and unloaded nanoparticles. It is concluded that the synthesized OESNPs with controlled release hold the potential to utilize industrial pea starch waste for the delivery of natural pesticides in agriculture.

Imparting Stability to Chiral Helical Gold Nanoparticle Superstructures.

Realizing the promise of chiral inorganic nanomaterials hinges on improving their structural stability under various chemical and environmental conditions. Here, we examine the stability of 1-D gold nanoparticle (Au NP) single helices prepared using the amphiphilic peptide conjugate Cx-(PEPAuM-ox)2 (PEPAuM-ox = AYSSGAPPMoxPPF; x = 16-22). We present a general template-independent strategy of tuning helix stability that relies on controlling the dimensions of constituent NPs. As NP dimensions increase, Au NP single helices become both more thermally stable and more stable in the presence of chemical denaturants and protein digestion agents (e.g., urea and proteinase K, respectively). We use this strategy for imparting helix stability to create colloidal suspensions of thermally robust Au NP single helices which maintain their plasmonic chiroptical activity up to ∼80 °C.

Fish antifreeze protein origin in sculpins by frameshifting within a duplicated housekeeping gene.

Antifreeze proteins (AFPs) are found in a variety of marine cold-water fishes where they prevent freezing by binding to nascent ice crystals. Their diversity (types I, II, III and antifreeze glycoproteins), as well as their scattered taxonomic distribution hint at their complex evolutionary history. In particular, type I AFPs appear to have arisen in response to the Late Cenozoic Ice Age that began ~ 34 million years ago via convergence in four different groups of fish that diverged from lineages lacking this AFP. The progenitor of the alanine-rich α-helical type I AFPs of sculpins has now been identified as lunapark, an integral membrane protein of the endoplasmic reticulum. Following gene duplication and loss of all but three of the 15 exons, the final exon, which encoded a glutamate- and glutamine-rich segment, was converted to an alanine-rich sequence by a combination of frameshifting and mutation. Subsequent gene duplications produced numerous isoforms falling into four distinct groups. The origin of the flounder type I AFP is quite different. Here, a small segment from the original antiviral protein gene was amplified and the rest of the coding sequence was lost, while the gene structure was largely retained. The independent origins of type I AFPs with up to 83% sequence identity in flounder and sculpin demonstrate strong convergent selection at the level of protein sequence for alanine-rich single alpha helices that bind to ice. Recent acquisition of these AFPs has allowed sculpins to occupy icy seawater niches with reduced competition and predation from other teleost species.

Self-assembly of curdlan molecules for the formation of thermally induced gels

The differences in the self-assembly process between cold-set (CCS) and heat-set (CHS) curdlan gels were investigated using micro-differential scanning calorimetry, atomic force microscopy, and small-angle X-ray diffraction. The results indicate that the triple-helix structure of curdlan dissociated into single helices in its suspension state (C-SUS) at 65 °C. Upon cooling to around 38 °C, these single helices assembled into long, thick fibers with a height of up to 11.50 nm, contributing to the gel structure of CCS. Heating C-SUS from 65 °C to 90 °C caused the single helices to transform into triple helices due to hydrophobic interactions. Subsequent cooling to 25 °C, led to the formation of short, thick bundles with a height of up to 8.78 nm, forming the network structure of CHS gel. As a consequence, CHS gels exhibited higher hardness and elasticity but lower fatigue resistance. This is attributed to an increased degree of physical cross-linking, as evidenced by a reduction in the size (Ξ) and spacing (ξ) of high-density domains within the gel structure, and a simpler mechanism of energy dissipation through alignment of the molecular chain segment. These findings are valuable for the design and development of curdlan-based gels with tailored mechanical properties.

Observation of edge states derived from topological helix chains.

Introducing the concept of topology has revolutionized materials classification, leading to the discovery of topological insulators and Dirac-Weyl semimetals1-3. One of the most fundamental theories underpinning topological materials is the Su-Schrieffer-Heeger (SSH) model4,5, which was developed in 1979-decades before the recognition of topological insulators-to describe conducting polymers. Distinct from the vast majority of known topological insulators with two and three dimensions1-3, the SSH model predicts a one-dimensional analogue of topological insulators, which hosts topological bound states at the endpoints of a chain4-8. To establish this unique and pivotal state, it is crucial to identify the low-energy excitations stemming from bound states, but this has remained unknown in solids because of the absence of suitable platforms. Here we report unusual electronic states that support the emergent bound states in elemental tellurium, the single helix of which was recently proposed to realize an extended version of the SSH chain9,10. Using spin- and angle-resolved photoemission spectroscopy with a micro-focused beam, we have shown spin-polarized in-gap states confined to the edges of the (0001) surface. Our densityfunctional theory calculations indicate that these states are attributed to the interacting bound states originating from the one-dimensional array of SSH tellurium chains. Helices in solids offer a promising experimental platform for investigating exotic properties associated with the SSH chain and exploring topological phases through dimensionality control.

Chiral and directional optical emission from a dipole source coupled to a helical plasmonic antenna

Plasmonic antennas with helical geometry can convert linearly polarized dipole radiation into purely circularly polarized far-fields, and vice versa. Besides large Purcell enhancements, they possess a wide tunability due to the geometry dependence of their resonant modes. Here, the coupling of a dipole emitter embedded in a thin film to plasmonic single and double helices is numerically studied. Using a higher-order finite element method (FEM), the wavelength dependent Purcell enhancement of a dipole with different positions and orientations is calculated and the far-fields with respect to their chirality and radiation patterns are analyzed. Both single and double helices demonstrate highly directional and circularly polarized far-fields for resonant excitation but with significantly improved directional radiation for the case of double helices.

Structural insights into the role of deleterious mutations at the dimeric interface of Toll-like receptor interferon-β related adaptor protein.

Toll-like receptors (TLRs) are major players in the innate immune system-recognizing pathogens and differentiating self/non-self components of immunity. These proteins are present either on the plasma membrane or endosome and recognize pathogens at their extracellular domains. They are characterized by a single transmembrane helix and an intracellular toll-interleukin-1 receptor (TIR) domain. Few TIRs directly invoke downstream signaling, while others require other TIR domains of adaptors like TIR domain-containing adaptor-inducing interferon-β (TRIF) and TRIF-related adaptor molecule (TRAM). On recognizing pathogenic lipopolysaccharides, TLR4 dimerises and interacts with the intracellular TRAM dimer through the TIR domain to recruit a downstream signaling adaptor (TRIF). We have performed an in-depth study of the structural effect of two mutations (P116H and C117H) at the dimeric interface of the adaptor TRAM, which are known to abrogate downstream signaling. We modeled the structure and performed molecular dynamics studies in order to decipher the structural basis of this effect. We observed that these mutations led to an increased radius of gyration of the complex and resulted in several changes to the interaction energy values when compared against the wild type (WT) and positive control mutants. We identified highly interacting residues as hubs in the WT dimer, and a few such hubs that were lost in the mutant dimers. Changes in the protein residue path, hampering the information flow between the crucial A86/E87/D88/D89 and T155/S156 sites, were observed for the mutants. Overall, we show that such residue changes can have subtle but long-distance effects, impacting the signaling path allosterically.

Proton-coupled transport mechanism of the efflux pump NorA.

Efflux pump antiporters confer drug resistance to bacteria by coupling proton import with the expulsion of antibiotics from the cytoplasm. Despite efforts there remains a lack of understanding as to how acid/base chemistry drives drug efflux. Here, we uncover the proton-coupling mechanism of the Staphylococcus aureus efflux pump NorA by elucidating structures in various protonation states of two essential acidic residues using cryo-EM. Protonation of Glu222 and Asp307 within the C-terminal domain stabilized the inward-occluded conformation by forming hydrogen bonds between the acidic residues and a single helix within the N-terminal domain responsible for occluding the substrate binding pocket. Remarkably, deprotonation of both Glu222 and Asp307 is needed to release interdomain tethering interactions, leading to opening of the pocket for antibiotic entry. Hence, the two acidic residues serve as a "belt and suspenders" protection mechanism to prevent simultaneous binding of protons and drug that enforce NorA coupling stoichiometry and confer antibiotic resistance.

Activation and Purification of ß-Glucocerebrosidase by Exploiting its Transporter LIMP-2 - Implications for Novel Treatment Strategies in Gaucher's and Parkinson's Disease.

Genetic variants of GBA1 can cause the lysosomal storage disorder Gaucher disease and are among the highest genetic risk factors for Parkinson's disease (PD). GBA1 encodes the lysosomal enzyme beta-glucocerebrosidase (GCase), which orchestrates the degradation of glucosylceramide (GluCer) in the lysosome. Recent studies have shown that GluCer accelerates α-synuclein aggregation, exposing GCase deficiency as a major risk factor in PD pathology and as a promising target for treatment. This study investigates the interaction of GCase and three disease-associated variants (p.E326K, p.N370S, p.L444P) with their transporter, the lysosomal integral membrane protein 2 (LIMP-2). Overexpression of LIMP-2 in HEK 293T cells boosts lysosomal abundance of wt, E326K, and N370S GCase and increases/rescues enzymatic activity of the wt and E326K variant. Using a novel purification approach, co-purification of untagged wt, E326K, and N370S GCase in complex with His-tagged LIMP-2 from cell supernatant of HEK 293F cells is achieved, confirming functional binding and trafficking for these variants. Furthermore, a single helix in the LIMP-2 ectodomain is exploited to design a lysosome-targeted peptide that enhances lysosomal GCase activity in PD patient-derived and control fibroblasts. These findings reveal LIMP-2 as an allosteric activator of GCase, suggesting a possible therapeutic potential of targeting this interaction.

Favored CH-π interaction between enzymatically modified high amylose starch and resveratrol improves digestion resistance

Starch-stilbene polyphenol complexes, a novel form of resistant starch (RS), have received considerable attention due to their potential health benefits. To enhance the functionality of this type of complex, we examined the interactions between resveratrol (RA) and high-amylose starch with different molecular weights, achieved through enzymatic modification followed by high-pressure homogenization (HPH). We examined how these interactions affect the digestibility and ordered structures of the complexes. Our findings reveal that, notwithstanding the inhibition of double helix and B-type crystalline structure formation, RA-starch complexation markedly boosted single helix content, V-type crystalline structure formation, and aggregate structure density. These effects contributed notably to increased RS content. Interestingly, the formation of the V6-type crystalline structure within the complexes was primarily facilitated by the CH-π interaction between the aromatic rings of RA and the C–H of starch, rather than conventional hydrogen bonding and electrostatic forces. The strength of the CH-π interaction increased with higher amylose content and lower molecular weight of the starch, achievable through a 12-h pullulanase hydrolysis followed by a 2-h α-amylase hydrolysis. The resulting HPH-Pα2-RA complexes exhibited remarkable levels of the V-type crystalline structure and RS content, reaching up to 25.45% and 57.37%, respectively. Overall, this study offers valuable insights into the design of starch-polyphenols complexes with a high RS content.

The role of C18 fatty acids in improving the digestion and retrogradation properties of highland barley starch

In this study, five C18 fatty acids (FA) with different numbers of double bonds and configurations including stearic acid (SA), oleic acid (OA), elaidic acid (EA), linoleic acid (LA), and α-linolenic acid (ALA), were selected to prepare highland barely starch (HBS)-FA complexes to modulate digestibility and elaborate the underlying mechanism. The results showed that HBS-SA had the highest complex index (34.18 %), relative crystallinity (17.62 %) and single helix content (25.78 %). Furthermore, the HBS-C18 FA complexes were formed by EA (C18 FA with monounsaturated bonds) that had the highest R1047/1022 (1.0509) and lowest full width at half-maximum (FWHM, 20.85), suggesting good short-range ordered structure. Moreover, all C18 FAs could form two kinds of V-type complexes with HBS, which can be confirmed by the results of CLSM and DSC measurements, and all of them showed significantly lower digestibility. HBS-EA possessed the highest resistant starch content (20.17 %), while HBS-SA had the highest slowly digestible starch content (26.61 %). In addition, the inhibition of HBS retrogradation by fatty acid addition was further proven, where HBS-SA gel firmness (37.80 g) and aging enthalpy value were the lowest, indicating the most effective. Overall, compounding with fatty acids, especially SA, could be used as a novel way to make functional foods based on HBS.

Identification of a novel Golgi-localized putative glycosyltransferase protein in Arabidopsis thaliana.

SNAREs play an important role in the process of membrane trafficking. In the present research, we investigated subcellular localization of an uncharacterized Arabidopsis thaliana protein reported to interact with a trans-Golgi network-localized Qa-SNARE, SYNTAXIN OF PLANTS 43. Based on the similarity of its amino acid sequence to metazoan fucosyltransferases, we have named this novel protein AtGTLP (Arabidopsis thaliana GlycosylTransferase-Like Protein) and predicted that it should be a member of yet uncharacterized family of Arabidopsis fucosyltransferases, as it shows no significant sequence similarity to fucosyltransferases previously identified in Arabidopsis. AtGTLP is a membrane-anchored protein, which exhibits a type II-like topology, with a single transmembrane helix and a globular domain in the C-terminal part of its amino acid sequence. Colocalization data we collected suggest that AtGTLP should localize mainly to Golgi apparatus, especially to certain zones of trans-Golgi. As single atgtlp-/- mutants showed no obvious difference in phenotype (primary root length and fresh mass), AtGTLP and proteins related to AtGTLP with high similarity in amino acid sequences may have redundant functions.