Membrane Domains Research Articles

The exocyst complex controls multiple events in the pathway of regulated exocytosis.

Eukaryotic cells depend on exocytosis to direct intracellularly synthesized material toward the extracellular space or the plasma membrane, so exocytosis constitutes a basic function for cellular homeostasis and communication between cells. The secretory pathway includes biogenesis of secretory granules (SGs), their maturation and fusion with the plasma membrane (exocytosis), resulting in release of SG content to the extracellular space. The larval salivary gland of Drosophila melanogaster is an excellent model for studying exocytosis. This gland synthesizes mucins that are packaged in SGs that sprout from the trans-Golgi network and then undergo a maturation process that involves homotypic fusion, condensation, and acidification. Finally, mature SGs are directed to the apical domain of the plasma membrane with which they fuse, releasing their content into the gland lumen. The exocyst is a hetero-octameric complex that participates in tethering of vesicles to the plasma membrane during constitutive exocytosis. By precise temperature-dependent gradual activation of the Gal4-UAS expression system, we have induced different levels of silencing of exocyst complex subunits, and identified three temporarily distinctive steps of the regulated exocytic pathway where the exocyst is critically required: SG biogenesis, SG maturation, and SG exocytosis. Our results shed light on previously unidentified functions of the exocyst along the exocytic pathway. We propose that the exocyst acts as a general tethering factor in various steps of this cellular process.

The exocyst complex controls multiple events in the pathway of regulated exocytosis

Eukaryotic cells depend on exocytosis to direct intracellularly synthesized material toward the extracellular space or the plasma membrane, so exocytosis constitutes a basic function for cellular homeostasis and communication between cells. The secretory pathway includes biogenesis of secretory granules (SGs), their maturation and fusion with the plasma membrane (exocytosis), resulting in release of SG content to the extracellular space. The larval salivary gland of Drosophila melanogaster is an excellent model for studying exocytosis. This gland synthesizes mucins that are packaged in SGs that sprout from the trans-Golgi network and then undergo a maturation process that involves homotypic fusion, condensation, and acidification. Finally, mature SGs are directed to the apical domain of the plasma membrane with which they fuse, releasing their content into the gland lumen. The exocyst is a hetero-octameric complex that participates in tethering of vesicles to the plasma membrane during constitutive exocytosis. By precise temperature-dependent gradual activation of the Gal4-UAS expression system, we have induced different levels of silencing of exocyst complex subunits, and identified three temporarily distinctive steps of the regulated exocytic pathway where the exocyst is critically required: SG biogenesis, SG maturation, and SG exocytosis. Our results shed light on previously unidentified functions of the exocyst along the exocytic pathway. We propose that the exocyst acts as a general tethering factor in various steps of this cellular process.

SARS-CoV-2 entry and fusion are independent of ACE2 localization to lipid rafts.

Membrane fusion occurs at the early stages of SARS-CoV-2 replication, during entry of the virus, and later during the formation of multinucleated cells called syncytia. Fusion is mediated by the binding of the viral Spike protein to its receptor ACE2. Lipid rafts are dynamic nanodomains enriched in cholesterol and sphingolipids. Rafts can act as platforms for entry of different viruses by localizing virus receptors, and attachment factors to the same membrane domains. Here, we first demonstrate that cholesterol depletion by methyl-beta-cyclodextrin inhibits Spike-mediated fusion and entry. To further study the role of ACE2 lipid raft localization in SARS-CoV-2 fusion and entry, we designed a GPI-anchored ACE2 construct. Both ACE2 and ACE2-GPI proteins were similarly expressed at the plasma membrane. Through membrane flotation assays, we show that in different cell lines, ACE2-GPI localizes predominantly to raft domains of the plasma membrane while ACE2 is non-raft associated. We then compare the ability of ACE2 and ACE2-GPI to permit SARS-CoV-2 entry, replication, and syncytia formation of different viral variants. We find little difference in the two proteins. Our results demonstrate that SARS-CoV-2 entry and fusion are cholesterol-dependent and raft-independent processes.IMPORTANCERafts are often exploited by viruses and used as platforms to enhance their entry into the cell or spread from cell to cell. The membrane localization of ACE2 and the role of lipid rafts in SARS-CoV-2 entry and cell-to-cell spread are poorly understood. The function of lipid rafts in viral fusion is often studied through their disruption by cholesterol-depleting agents. However, this process may have off-target impacts on viral fusion independently of lipid-raft disruption. Therefore, we created an ACE2 construct that localizes to lipid rafts using a GPI anchor. Conversely, wild-type ACE2 was non-raft associated. We find that the localization of ACE2 to lipid rafts does not modify the fusion dynamics of SARS-CoV-2.

Membrane lipid nanodomains modulate HCN pacemaker channels in nociceptor DRG neurons

Cell membranes consist of heterogeneous lipid nanodomains that influence key cellular processes. Using FRET-based fluorescent assays and fluorescence lifetime imaging microscopy (FLIM), we find that the dimension of cholesterol-enriched ordered membrane domains (OMD) varies considerably, depending on specific cell types. Particularly, nociceptor dorsal root ganglion (DRG) neurons exhibit large OMDs. Disruption of OMDs potentiated action potential firing in nociceptor DRG neurons and facilitated the opening of native hyperpolarization-activated cyclic nucleotide-gated (HCN) pacemaker channels. This increased neuronal firing is partially due to an increased open probability and altered gating kinetics of HCN channels. The gating effect on HCN channels is likely due to a direct modulation of their voltage sensors by OMDs. In animal models of neuropathic pain, we observe reduced OMD size and a loss of HCN channel localization within OMDs. Additionally, cholesterol supplementation inhibited HCN channels and reduced neuronal hyperexcitability in pain models. These findings suggest that disturbances in lipid nanodomains play a critical role in regulating HCN channels within nociceptor DRG neurons, influencing pain modulation.

Proximity labeling reveals interactions necessary to maintain the distinct apical domains of Drosophila photoreceptors.

Specialized membrane and cortical protein regions are common features of cells and are utilized to isolate differential cellular functions. In Drosophila photoreceptors, the apical membrane domain is defined by two distinct morphological membranes: the rhabdomere microvilli and the stalk membrane. To define the apical cortical protein complexes, we performed proximity labeling screens utilizing the rhabdomeric-specific protein PIP82 as bait. We found that the PIP82 interactome is enriched for actin-binding and cytoskeleton proteins, as well as proteins for cellular trafficking. Analysis of one target, Bifocal, with PIP82 revealed two independent pathways for localization to the rhabdomeric membrane and an additional mechanism of crosstalk between the protein complexes of the rhabdomeric and stalk membranes. The loss of Bifocal, and enhancement in the Bifocal/PIP82 double mutant, results in the additional distribution of Crumbs, an apical stalk membrane protein, to the lateral basal photoreceptor membrane. This phenotype is recapitulated by the knockdown of the catalytic subunit of Protein Phosphatase 1, a known interactor of Bifocal. Taken together, these results expand our understanding of the molecular mechanisms underlying the generation of the two distinct photoreceptor apical domains.

In vitro characteristics of purified recombinant hepatitis C virus core protein

In our previous study, we established a method for purifying bacterially expressed HCV core 1–164 under non-denaturing conditions. In the present study, we elucidated the characteristics of the purified core. The purified HCV core exhibited a notable affinity for HCV RNA, with a Kd of 3 nM, as determined by a filter binding assay. Electron microscopy analysis revealed that the purified HCV core self-assembled with RNA into spherical structures approximately 50 nm in diameter. Additionally, we demonstrated the direct binding of the purified HCV core to the purified endoplasmic reticulum (ER). Moreover, lipid strip assays revealed specific binding of the purified HCV core to specific phospholipids, suggesting that the core plays a role in specific ER membrane domains. These studies reveal the biochemical characteristics of the HCV core that significantly advance our understanding of its role as a capsid protein in the viral lifecycle and pathogenesis.

Detection of Androgen Receptors in Spermatozoa of Small Ruminants: A Putative Modulation Pathway for Cryoresistance Through AQP3.

This work was aimed to identify androgen receptors (AR) in the spermatozoa of wild and domestic ruminants and to assess the effect of testosterone on sperm localization of aquaporin-3 (AQP3) and cryopreservation process. Sperm samples from wild species were incubated with testosterone (T group), 1,3-propanediol (PDO group), phloretin (PHL group), PDO+T group, PHL+T group. Western blot identified the presence of AR as a single band of about 48 KDa. Immunolabelling of AR was located in the equatorial segment of the sperm head. In mouflons, the cryoresistance ratio for acrosome integrity was lower (p < 0.05) in the PHL+T than in Control and T groups. In ibexes, the cryoresistance ratio for acrosome integrity was lower (p < 0.05) in the PHL+T, PHL, and T group than in the Control group; the cryoresistance ratios for sperm kinematic variables were lower (p < 0.05) in PDO+T than in Control. No changes were found among treatments in the proportion of spermatozoa showing AQP3 in the different membrane domains after incubation and thawing in both mouflon and ibex. In conclusion, testosterone negatively affected sperm cryoresistance expressed as acrosome integrity, enhancing the effects of the AQP blocker PHL. Our findings provide a sound knowledge of the molecular mechanisms that explain the seasonal variation in sperm freezability from ruminants.

The transmembrane domain of the desmosomal cadherin desmoglein-1 governs lipid raft association to promote desmosome adhesive strength.

Cholesterol- and sphingolipid-enriched domains called lipid rafts are hypothesized to selectively coordinate protein complex assembly within the plasma membrane to regulate cellular functions. Desmosomes are mechanically resilient adhesive junctions that associate with lipid raft membrane domains, yet the mechanisms directing raft association of the desmosomal proteins, particularly the transmembrane desmosomal cadherins, are poorly understood. We identified the desmoglein-1 (DSG1) transmembrane domain (TMD) as a key determinant of desmoglein lipid raft association and designed a panel of DSG1TMD variants to assess the contribution of TMD physicochemical properties (length, bulkiness, and palmitoylation) to DSG1 lipid raft association. Sucrose gradient fractionations revealed that TMD length and bulkiness, but not palmitoylation, govern DSG1 lipid raft association. Further, DSG1 raft association determines plakoglobin recruitment to raft domains. Super-resolution imaging and functional assays uncovered a strong relationship between the efficiency of DSG1TMD lipid raft association and the formation of morphologically and functionally robust desmosomes. Lipid raft association regulated both desmosome assembly dynamics and DSG1 cell surface stability, indicating that DSG1 lipid raft association is required for both desmosome formation and maintenance. These studies identify the biophysical properties of desmoglein transmembrane domains as key determinants of lipid raft association and desmosome adhesive function.

The Role of Cholesterol in M2 Clustering and Viral Budding Explained.

The influenza A M2 homotetrameric channel consists of four transmembrane (TM) and four amphipathic helices (AHs). This viral proton channel is suggested to form clusters in the catenoid budding neck areas in raft-like domains of the plasma membrane, resulting in cell membrane scission and viral release. The channel clustering environment is rich in cholesterol. Previous experiments have shown that cholesterol significantly contributes to lipid bilayer undulations in viral buds. However, a clear explanation of membrane curvature from the distribution of cholesterol around the M2TM-AH clusters is lacking. Using coarse-grained molecular dynamics simulations of M2TM-AH in bilayers, we observed that M2 channels form specific, C2-symmetric, clusters with conical shapes driven by the attraction of their AHs. We showed that cholesterol stabilized the formation of M2 channel clusters by filling and bridging the conical gap between M2 channels at specific sites in the N-termini of adjacent channels or via the C-terminal region of TM and AHs, with the latter sites displaying a longer interaction time and higher stability. The potential of mean force calculations showed that when cholesterols occupy the identified interfacial binding sites between two M2 channels, the dimer is stabilized by 11 kJ/mol. This translates to the cholesterol-bound dimer being populated by almost 2 orders of magnitude compared to a dimer lacking cholesterol. We demonstrated that the cholesterol-bridged M2 channels can exert a lateral force on the surrounding membrane to induce the necessary negative Gaussian curvature profile, which permits spontaneous scission of the catenoid membrane neck and leads to viral buds and scission.

Exploring Free Energy Landscapes for Protein Partitioning into Membrane Domains in All-Atom and Coarse-Grained Simulations.

It is known that membrane environment can impact the structure and function of integral membrane proteins. As such, elucidation of the thermodynamic driving forces governing protein partitioning between membrane domains of varying lipid composition is a fundamental topic in membrane biophysics. Molecular dynamics simulations provide valuable tools for quantitatively characterizing the free energy landscapes governing protein partitioning at the molecular level. In this study, we propose an efficient simulation methodology for the calculation of free energies for the partitioning of transmembrane proteins between liquid-disorder (Ld) and liquid-ordered (Lo) domains in all-atom (AA) phase-separated lipid bilayers. The computed potential of mean force defining the equilibrium partition coefficients is compared for AA and coarse-grained systems. Energy decomposition is used to identify differences in the underlying thermodynamics. Our findings highlight the importance of employing AA models to accurately estimate relevant free energy changes during protein translation between membrane domains.

The Effects of Viral Structural Proteins on Acidic Phospholipids in Host Membranes.

Enveloped viruses rely on host membranes for trafficking and assembly. A substantial body of literature published over the years supports the involvement of cellular membrane lipids in the enveloped virus assembly processes. In particular, the knowledge regarding the relationship between viral structural proteins and acidic phospholipids has been steadily increasing in recent years. In this review, we will briefly review the cellular functions of plasma membrane-associated acidic phospholipids and the mechanisms that regulate their local distribution within this membrane. We will then explore the interplay between viruses and the plasma membrane acidic phospholipids in the context of the assembly process for two enveloped viruses, the influenza A virus (IAV) and the human immunodeficiency virus type 1 (HIV-1). Among the proteins encoded by these viruses, three viral structural proteins, IAV hemagglutinin (HA), IAV matrix protein-1 (M1), and HIV-1 Gag protein, are known to interact with acidic phospholipids, phosphatidylserine and/or phosphatidylinositol (4,5)-bisphosphate. These interactions regulate the localization of the viral proteins to and/or within the plasma membrane and likely facilitate the clustering of the proteins. On the other hand, these viral proteins, via their ability to multimerize, can also alter the distribution of the lipids and may induce acidic-lipid-enriched membrane domains. We will discuss the potential significance of these interactions in the virus assembly process and the property of the progeny virions. Finally, we will outline key outstanding questions that need to be answered for a better understanding of the relationships between enveloped virus assembly and acidic phospholipids.

The troubles with the membrane domains

The troubles with the membrane domains

Genome-wide identification and expression analysis of CASPL gene family in Zea mays (L.).

Casparian strip membrane domain proteins like (CASPL), exhibit profound associations with root development, stress responsiveness and mineral element uptake in plants. Nonetheless, a comprehensive bioinformatics analysis of the ZmCASPL gene family in maize remains unreported. In the study, we have identified 47 ZmCASPL members at the whole-genome level, systematically classifying them into six distinct groups. Furthermore, our analysis revealed that the same group of ZmCASPL contains similar gene structures and conserved motifs. Duplication events showed whole genome duplication (WGD) and tandem duplication (TD) contribute to the generation of the ZmCASPL gene family together in maize, but the former plays a more prominent role. Furthermore, we observed that most ZmCASPL genes contain MYB-binding sites (CAACCA), which are associated with the Casparian strip. Utilizing RNA-seq data, we found that ZmCASPL21 and ZmCASPL47 are specifically highly expressed only in the roots. This finding implies that ZmCASPL21 and ZmCASPL47 may be involved in the Casparian strip development. Additionally, RNA-seq analysis illuminated that drought, salt, heat, cold stresses, low nitrogen and phosphorus conditions, as well as pathogen infection, significantly impact the expression patterns of ZmCASPL genes. RT-qPCR revealed that ZmCASPL 5/13/25/44 genes showed different expression patterns under PEG and NaCl treatments. Collectively, these findings provide a robust theoretical foundation for further investigations into the functional roles of the ZmCASPL gene family in maize.

Plasma membrane and cytoplasmic compartmentalization: a dynamic structural framework required for pollen tube tip growth.

Rapid, unidirectional pollen tube tip growth is essential for fertilization and is widely employed as a model of polar cell expansion, a process crucial for plant morphogenesis. Different proteins and lipids with key functions in the control of polar cell expansion are associated with distinct domains of the plasma membrane (PM) at the pollen tube tip. These domains need to be dynamically maintained during tip growth, which depends on massive secretory and endocytic membrane trafficking. Very little is currently known about the molecular and cellular mechanisms responsible for the compartmentalization of the pollen tube PM. To provide a reliable structural framework for the further characterization of these mechanisms, an integrated quantitative map was compiled of the relative positions in normally growing Nicotiana tabacum (tobacco) pollen tubes of PM domains 1) enriched in key signaling proteins or lipids, 2) displaying high membrane order, or 3) in contact with cytoplasmic structures playing important roles in apical membrane trafficking. Previously identified secretory and endocytic PM domains were also included into this map. Internalization of regulatory proteins or lipids associated with PM regions overlapping with the lateral endocytic domain was assessed based on brefeldin A (BFA) treatment. These analyses revealed remarkable aspects of the structural organization of tobacco pollen tube tips, which 1) enhance our understanding of cellular and regulatory processes underlying tip growth, and 2) highlight important areas of future research.

Proteomic and Targeted Lipidomic Analyses of Fluid and Rigid Rubber Particle Membrane Domains in Guayule.

Rubber (cis-1,4-polyisoprene) is produced in cytosolic unilamellar vesicles called rubber particles (RPs), and the protein complex responsible for this synthesis, the rubber transferase (RTase), is embedded in, or tethered to, the membranes of these RPs. Solubilized enzyme activity is very difficult to achieve because the polymerization of highly hydrophilic substrates into hydrophobic polymers requires a polar/non-polar interface and a hydrophobic compartment. Using guayule (Parthenium argentatum) as a model rubber-producing species, we optimized methods to isolate RP unilamellear membranes and then a subset of membrane microdomains (detergent-resistant membranes) likely to contain protein complexes such as RTase. The phospholipid and sterol composition of these membranes and microdomains were analyzed using thin-layer chromatography (TLC) and liquid chromatography tandem mass spectroscopy (LC-MS/MS). Our data indicate that RP membranes consist predominantly of phosphatidic acid-containing membrane microdomains (DRMs or "lipid rafts"). Proteomic analyses of guayule RP membranes and membrane microdomains identified 80 putative membrane proteins covering 30 functional categories. From this population, we have tentatively identified several proteins in multiple functional domains associated with membrane microdomains which may be critical to RTase function. Definition of the mechanisms underlying rubber synthesis will provide targets for both metabolic engineering and breeding strategies designed to increase natural rubber production in latex-producing species.

Implications of the Lipidic Ecosystem for the Membrane Binding of ApoE Signal Peptide: Importance of Sphingomyelin.

The unidirectional movement of nascent secretory proteins in the cell is primarily assisted by the signal recognition particles (SRP). However, this does not completely justify the importance of the signal peptide (SP) which gets eliminated after the protein translocation. We have earlier demonstrated that a negatively charged lipid such as POPG plays an important role in the higher binding affinity and cholesterol-discriminating ability of the apolipoprotein E (ApoE) SP. In this present work, we aimed to understand the role of sphingomyelin, an important constituent of ER, on the membrane binding of ApoE SP. Our results demonstrate that sphingomyelin promotes membrane binding but cannot discriminate cholesterol. However, sphingomyelin shows a synergistic effect with POPG toward the membrane binding of the ApoE SP. We have further shown that the membrane domains do not have any impact on the binding of ApoE SP. Based on our results we propose that the lipid composition of the endoplasmic reticulum (ER) where ApoE translocates, enhances the binding of the ApoE signal peptide to the ER membrane.

Lipid Compositions of Liquid-Ordered and Liquid-Disordered Phases in Ternary Membranes of Sphingomyelin, Cholesterol, and Dioleoylphosphatidylcholine Determined by 2H NMR: Stearoyl-Sphingomyelin Compared with Its Palmitoyl Counterpart.

Sphingomyelin (SM) and cholesterol are the major lipids in the signaling platforms of cell membranes, known as lipid rafts. In particular, SM with a stearoyl chain (C18-SM) is abundant in specific tissues such as the brain, the most cholesterol-rich organ, whereas the distribution of palmitoyl (C16)-SM is ubiquitous. Here, we reveal the differences between palmitoyl- and stearoyl-SM in lipid-lipid interactions based on the tie lines obtained from the 2H solid-state NMR spectra of bilayer systems composed of SM/dioleoylphosphatidylcholine/cholesterol 33:33:33 and 40:40:20. Lipid probes carrying position-selective deuterations, 10',10'-d2-SM, 24-d1-cholesterol, and 6″,6″-d2-dioleoyl-phosphatidylcholine, were incorporated into the membranes. 2H NMR peaks from these probes in the membranes directly provide the lipid compositions of the liquid-ordered (Lo) and liquid-disordered (Ld) regions. Without using bulky fluorescent groups, these probes allow us to obtain the end points of the tie lines in a ternary phase diagram based on the lever rule. Consequently, the tie lines of the stearoyl-SM membranes were steeper than those of the palmitoyl-SM membranes, indicating that cholesterol content was higher in the Lo domains of stearoyl-SM, regardless of the total concentration of unsaturated phospholipids. When comparing the content of unsaturated lipids in the Lo domain, the stearoyl-SM membranes had a higher content than palmitoyl-SM membranes. These results revealed that stearoyl-SM is suitable for stabilizing biologically functional microdomains in cholesterol-rich organs, whereas palmitoyl-SM may be better suited for stabilizing domains in tissue membranes with normal cholesterol content. The small but significant differences in the lipid interactions between stearoyl-SM and palmitoyl-SM may be related to the spatiotemporal formation of functional domains in biological environments.

The Multifaceted Role of Epithelial Membrane Protein 2 in Cancer: from Biomarker to Therapeutic Target.

Tetraspanin superfamily proteins not only facilitate the trafficking of specific proteins to distinct plasma membrane domains but also influence cell-to-cell and cell-extracellular matrix interactions. Among these proteins, Epithelial Membrane Protein 2 (EMP2), a member of the growth arrest-specific gene 3/peripheral myelin protein 22 (GAS3/PMP22) family, is known to affect key cellular processes. Recent studies have revealed that EMP2 modulates critical signaling pathways and interacts with adhesion molecules and growth factor receptors, underscoring its potential as a biomarker for cancer diagnosis and prognosis. These findings suggest that EMP2 expression patterns could provide valuable insights into tumorigenesis and metastasis. Moreover, EMP2 has emerged as a promising therapeutic target, with approaches aimed at inhibiting or modulating its activity showing potential to disrupt tumor growth and metastasis. This review provides a comprehensive overview of recent advances in understanding the multifaceted roles of EMP2 in cancer, with a focus on its underlying mechanisms and clinical significance.

The GTPase RAB6 is required for stem cell maintenance and cell migration in the gut epithelium.

Intestinal epithelial cells, which are instrumental in nutrient absorption, fluid regulation, and pathogen defense, undergo continuous proliferation and differentiation within the intestinal crypts, migrating towards the luminal surface where they are eventually shed. RAB GTPases are key regulators of intracellular vesicular trafficking and are involved in various cellular processes, including cell migration and polarity. Here, we investigated the role of RAB6 in the development and maintenance of the gut epithelium. We generated conditional knockout mice with RAB6 specifically deleted in the gut epithelium. We found that deletion of the Rab6a gene resulted in embryonic lethality. In adult mice, RAB6 depletion led to altered villus architecture and impaired junction integrity without affecting the segregation of apical and basolateral membrane domains. Further, RAB6 depletion slowed down cell migration and adversely affected both cell proliferation and stem cell maintenance. Notably, the absence of RAB6 resulted in a diminished number of functional stem cells, as evidenced by the rapid death of isolated crypts from Rab6a KO mice when cultured as 3D organoids. Together, these results underscore the essential role of RAB6 in maintaining gut epithelial homeostasis.

IQGAP-2: a novel interacting partner with the human colonic thiamin pyrophosphate transporter.

The human colonic thiamin pyrophosphate transporter (hcTPPT) mediates the uptake of the microbiota-generated and phosphorylated form of vitamin B1 (i.e., thiamin pyrophosphate) in the large intestine. Expression of hcTPPT along the absorptive tract is restricted to the large intestine, and the transporter is exclusively localized at the apical membrane domain of the polarized epithelial cells/colonocytes. Previous studies have characterized different physiological/pathophysiological aspects of the hcTPPT system, but nothing is currently known on whether the transporter has interacting partner(s) that affect its physiology/biology. We addressed this issue using a Y2H to screen a human colonic cDNA library and have identified three putative interactors, namely IQGAP-2, SNX-6, and DMXL-1. Focusing on IQGAP-2 (whose expression in human colonocytes is the highest), we found (using fluorescent microscopy imaging and coimmunoprecipitation approaches) the putative interactor to colocalize with hcTPPT and to directly interact with the transporter. Also, overexpressing IQGAP-2 in NCM460 cells and in human primary differentiated colonoid monolayers was found to lead to significant (P < 0.01) induction in TPP uptake, while knocking down (using gene-specific siRNAs) caused significant (P < 0.01 and P < 0.05) decrease in uptake. Furthermore, overexpressing IQGAP-2 in NCM460 cells was found to lead to a significant enhancement in hcTPPT protein stability. Finally, we found the expression of IQGAP-2 to be markedly suppressed in conditions/factors that negatively impact colonic TPP uptake. These results identify the IQGAP-2 as an interacting partner with the hcTPPT in human colonocytes and show that this interaction has physiological and biological consequences.NEW & NOTEWORTHY This study reports on the identification of IQGAP-2 as an interacting partner with the hcTPPT in human colonocytes and how that impacts the transporter's physiology and cell biology.