Maintenance Of Cellular Polarity Research Articles

Comprehensive analysis of glycerolipid dynamics during tobacco pollen germination and pollen tube growth.

Pollen germination and subsequent pollen tube elongation are essential for successful land plant reproduction. These processes are achieved through well-documented activation of membrane trafficking and cell metabolism. Despite this, our knowledge of the dynamics of cellular phospholipids remains scarce. Here we present the turnover of the glycerolipid composition during the establishment of cell polarity and elongation processes in tobacco pollen and show the lipid composition of pollen plasma membrane-enriched fraction for the first time. To achieve this, we have combined several techniques, such as lipidomics, plasma membrane isolation, and live-cell microscopy, and performed a study with different time points during the pollen germination and pollen tube growth. Our results showed that tobacco pollen tubes undergo substantial changes in their whole-cell lipid composition during the pollen germination and growth, finding differences in most of the glycerolipids analyzed. Notably, while lysophospholipid levels decrease during germination and growth, phosphatidic acid increases significantly at cell polarity establishment and continues with similar abundance in cell elongation. We corroborated these findings by measuring several phospholipase activities in situ. We also observed that lysophospholipids and phosphatidic acid are more abundant in the plasma membrane-enriched fraction than that in the whole cell. Our results support the important role for the phosphatidic acid in the establishment and maintenance of cellular polarity in tobacco pollen tubes and indicate that plasma membrane lysophospholipids may be involved in pollen germination.

Microtubule organization across cell types and states

Encircling and traversing the cell are architectural struts and dynamic intracellular highways made of cylindrical polymers called microtubules. Built from structurally asymmetric subunits of αβ-tubulin heterodimers, microtubules have an inherent structural polarity with a slow-growing minus end and a comparatively dynamic plus end that grows and shrinks. Thus, a key feature of microtubules is that each polymer is polarized, allowing for the execution of cellular tasks that are directional in nature. For example, microtubules build polarized highways allowing directional intracellular transport, generate directional force such as in chromosome alignment and segregation, provide structural support for cell shape, and assemble into highly ordered polar structures like centrioles and cilia. The output of these microtubule-based functions is the performance of different tasks, including establishment and maintenance of cellular polarity, secretion and absorption, cell-cell communication, migration, mechanical resiliency, and mitosis. Different cells accomplish these functions by using distinct sites within the cell called microtubule-organizing centers (MTOCs) to build cell-specific microtubule arrangements. While the specific requirement for microtubules in many in vivo cell types is unknown, disrupting even a subset of microtubule-supported functions is often lethal and is associated with many diseases (e.g., cancer and neuropathies), suggesting that specific patterns of microtubule organization are likely important for cellular function in vivo. This Primer focuses on how differentiated animal and plant cells use distinct MTOCs to generate specific microtubule arrangements, how those arrangements support cellular functions, and how cells rearrange their microtubules to accommodate changing cellular tasks.

Bioenergetics for Hepatocyte Polarization: Coordination of Multiple Cellular Organelles and the Master Regulator AMPK.

Hepatocytes have well-defined polarized morphological structure. Establishment and maintenance of cellular polarity is crucial to the function and viability of hepatocytes. Many disorders, both genetic and acquired, and drugs can impair hepatocyte polarization resulting in liver dysfunction and injury. Hepatocyte polarization is an energy-dependent cellular process. Hence, cellular energy metabolism can significantly affect the polarized morphology and function of hepatocytes. Recent in vitro studies have demonstrated that multiple cellular organelles, including mitochondria, auto-phagosomes and lipid droplets, are involved in the utilization of energy for hepatocyte polarization; also, AMP-dependent kinase (AMPK), a key cellular energy sensor, regulates the function and activities of these cellular organelles and thus plays an important role in bioenergetics for hepatocyte polarization. This review provides the latest understanding on how mitochondria, autophagosomes, and lipid droplets coordinate energy production, and it addresses how AMPK activation regulates these key cellular organelles for energy production during hepatocyte polarization. Furthermore, the review suggests potential directions for future research.

Regulation of Cell Polarity by Exocyst-Mediated Trafficking.

One requirement for establishing polarity within a cell is the asymmetric trafficking of intracellular vesicles to the plasma membrane. This tightly regulated process creates spatial and temporal differences in both plasma membrane composition and the membrane-associated proteome. Asymmetric membrane trafficking is also a critical mechanism to regulate cell differentiation, signaling, and physiology. Many eukaryotic cell types use the eight-protein exocyst complex to orchestrate polarized vesicle trafficking to certain membrane locales. Members of the exocyst were originally discovered in yeast while screening for proteins required for the delivery of secretory vesicles to the budding daughter cell. The same eight exocyst genes are conserved in mammals, in which the specifics of exocyst-mediated trafficking are highly cell-type-dependent. Some exocyst members bind to certain Rab GTPases on intracellular vesicles, whereas others localize to the plasma membrane at the site of exocytosis. Assembly of the exocyst holocomplex is responsible for tethering these vesicles to the plasma membrane before their soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-mediated exocytosis. In this review, we will focus on the role and regulation of the exocyst complex in targeted vesicular trafficking as related to the establishment and maintenance of cellular polarity. We will contrast exocyst function in apicobasal epithelial polarity versus front-back mesenchymal polarity, and the dynamic regulation of exocyst-mediated trafficking during cell phenotype transitions.

Rapid Isolation of Total Protein from Arabidopsis Pollen.

Arabidopsis pollen is an excellent system for answering important biological questions about the establishment and maintenance of cellular polarity and polar cell growth, because these processes are amenable to the genetic and genomic approaches that are readily available in Arabidopsis. Given that proteins are the direct executors of a wide variety of cellular processes, it is important to rapidly and efficiently isolate total protein for various protein-based analyses, such as Western blotting, co-immunoprecipitation and mass spectrometry, among others. Here we present a protocol for rapid isolation of total protein from Arabidopsis pollen, which is adapted from our recently published paper (Chang and Huang, 2015).

Rab25 in cancer: a brief update

Derailed endocytosis is a hallmark of cancer. The endocytic pathway, as demonstrated by our laboratory, is a frequent target of genomic aberrations in cancer and plays a critical role in the maintenance of cellular polarity, stem cell function, bioenergetics, proliferation, motility, invasion, metastasis, apoptosis and autophagy. The Rab GTPases, along with their effectors, are critical regulators of this endocytic machinery and can have a huge impact on the cellular itinerary of growth and metabolism. Rab25 is an epithelial-cell-specific member of the Rab GTPase superfamily, sharing close homology with Rab11a, the endosomal recycling Rab GTPase. RAB25 has been implicated in various cancers, with reports presenting it as both an oncogene and a tumour-suppressor gene. At the cellular level, Rab25 was shown to contribute to invasiveness of cancer cells by regulating integrin trafficking. Recently, our laboratory uncovered a critical role for Rab25 in cellular energetics. Assimilating all of the existing evidence, in the present review, we give an updated overview of the complex and often context-dependent role of Rab25 in cancer.

P4-05-01: Rab25 Alters Cellular Energetics and Growth Signaling during Breast Oncogenesis.

Abstract Background: Conservation and consumption of cellular energy is centrally regulated by growth and metabolism of the whole organism. The cancer cell gains autonomy from this regulation to become an organism with indeterminate growth potential. Plasma membrane receptors including growth factors and nutrient receptors are sensors for microenvironmental cues that dictate the intracellular energetic pathways to recalibrate ATP production based on available resources. So altercations in both growth and metabolic pathways are common features of cancers. A unifying component between receptor mediated growth signaling and metabolic pathways within a cell could be the vesicular trafficking system that imports, transports and exports a wide array of cargo from both these pathways. In fact, derailed endocytosis is a hallmark of cancer. The endocytic pathway, as demonstrated by our laboratory, is a frequent target of genomic aberrations in cancer, and plays a critical role in maintenance of cellular polarity, stem cell fnction, bioenergetics, proliferation, motility, invasion, metastasis, apoptosis and autophagy. The Rab GTPases along with their effectors are critical regulators of this endocytic machinary and can have a huge impact on the cellular iterinary of growth and metabolism. Methods and results: A broad spectrum analysis of distribution of all the Rab family members across 52 breast cancer cell lines reflected a dichotomous distribution of Rab25, with higher mRNA and protein expression in ER+ cell lines. Rab 25 is of immense interest to us because of its unique substitution of leucine for glutamine at position 71which typically decreases the intrinsic GTPase activity, resulting in a dominant, constitutively active protein, akin to a transforming RAS mutation. Using high throughput mRNA data analysis, we report that Rab25 and its effector Rab Coupling Protein (RCP) are frequently coamplified and coordinately elevated in ER +ve breast cancers. Specifically in the luminal B subtypes, they are significantly associated with a markedly worsened outcome. In vitro, in MCF7 cell line, we observe an increased EGFR stability and MAPK signaling in Rab25 overexpressing stable clones which could be reversed with shRNA knockdown of Rab25. Loss of either Rab25 or RCP inhibited survival and growth of MCF7 lines under serum free conditions suggesting that Rab25 can potentially facilitate growth factor independence in cancer cells. Most importantly, we report that Rab25 enhances mitochondrial ATP production in breast and ***ovarian cancer cells and significantly reduces acidification of the ECM. Our ongoing experiments are directed towards understanding the mechanistic link between Rab25 mediated effects on EGFR/pERK signaling and its effects on cellular metabolism. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-05-01.

RalA and RalB differentially regulate development of epithelial tight junctions

Tight junctions (TJs) are structures indispensable to epithelial cells and are responsible for regulation of paracellular diffusion and maintenance of cellular polarity. Although many interactions between TJ constituents have been identified, questions remain concerning how specific functions of TJs are established and regulated. Here we investigated the roles of Ral GTPases and their common effector exocyst complex in the formation of nascent TJs. Unexpectedly, RNA interference-mediated suppression of RalA or RalB caused opposing changes in TJ development. RalA reduction increased paracellular permeability and decreased incorporation of components into TJs, whereas RalB reduction decreased paracellular permeability and increased incorporation of components into TJs. Activities of both Ral GTPases were mediated through the exocyst. Finally, we show that TJ-mediated separation of apical-basal membrane domains is established prior to equilibration of barrier function and that it is unaffected by Ral knockdown or specific composition of TJs.

Abstract P4-04-04: Polarity Abnormalities in Invasive Carcinomas of the Breast: Analysis of Invasive Micropapillary Carcinoma of the Breast

Abstract Aims: Invasive micropapillary carcinomas (IMPC) are rare (<2% of all invasive breast carcinomas) and characterized by clusters of cells with an inverted polarity surrounded by a clear space that separate them from the extracellular matrix. These tumors harbor vascular invasion and axillary lymph node involvement in more than 70% of the cases. We performed a detailed analysis of cellular polarity in a series of 25 IMPC to get insight in the putative role of proteins involved in the maintenance of cellular polarity in that carcinoma subtype. Methods: Polarity analysis has been performed by immunohistochemistry on a tissue-micro-array of a series of twenty-five invasive micropapillary carcinomas. Apical (p-ERM, a marker of phosphorylated ezrin, moesin and radixin, aPKCz, MUC1 and GM130, a marker of the golgi apparatus), basal (collagen IV), or baso-lateral (EGFR, E-cadherin, b-catenin and ERBB2) markers were assessed. Results: Apical markers MUC1, aPKCz, P-ERM were expressed at the external cellular pole in 88% (22/25), 60% (15/25) and 40% (10/25) of the cases respectively. Notably, 15/25 (60%) were p-ERM negative. Twenty-four cases/25 (96%) demonstrated a GM130 localisation at the external pole of the cells, with an abnormal internal and external localisation of the labelling in 15/24 positive cases (62%). Finally, 24% (6/25) cases presented all these apical markers located at the external part of the cells and 92% (23/25) had at least one of the apical markers in external position. The large majority of the cases (96%; 24/25) demonstrated no collagen IV labelling at the external part of the cell clusters. Intercellular junction proteins E-cadherin and b-catenin were expressed in all cases with a complete absence of staining at the apical inverted pole for both markers. Interestingly, a thick and strong labelling was observed for E-cadherin and for b-catenin in 36% (9/25) and 92% (23/25) of the cases respectively. EGFR was negative in 24 out of the 25 cases (96%). In contrast, ERBB2 was overexpressed in 6/25 (24%) of cases with a membranous ERBB2 labelling except external at the inverted apical pole. EGFR and ERBB2 are considered in normal cells as a baso-lateral marker. Conclusion: IMPC present a clear inverted apical pole MUC-1 and/or aPKCz and/or phospho-ERM positive with the golgi apparatus facing the external membrane of cell clusters, oriented towards the clear space and the extracellular matrix. The intercellular junctions in IMPC are abnormal with a thick E-cadherin and b-catenin labelling compared to normal cells. These abnormal cell orientation and junctions could be a consequence of molecular alterations that are currently being analysed in our laboratory, and that could explain the high frequency of loco-regional lymphatic tumor extension observed in IMPC. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-04-04.

Genomic and proteomic approaches identify Rab25 as a potential prognostic marker for luminal B breast tumors

Abstract Estrogen receptor (ER) +ve luminal B subtype of breast tumors initially respond well to tamoxifen treatment. However, over longer periods of time, their frequent recurrence contributes to the overall high mortality rate of hormone receptor +ve breast cancer patients. Recently, with the development of PARP inhibitors, there is a potential line of treatment for the triple negative subtype. But a huge dearth exists in therapeutically targeting luminal B tumors since these tumors are also insensitive to chemotherapy. Our current research addresses this urgent need by utilizing a systems biology approach to identify potential therapeutic targets and diagnostic markers for luminal B subtype of breast cancer. The endocytic pathway, as demonstrated by our laboratory is a frequent target of genomic aberrations in cancer, and plays a critical role in maintenance of cellular polarity, stem cell function, bioenergetics, proliferation, motility, invasion, metastasis, apoptosis and autophagy. Derailed endocytosis is therefore a hallmark of cancer. The Rab GTPases along with their effectors are critical regulators of this endocytic machinery and can have a huge impact on the cellular itinerary of growth factor as well as integrin receptor signaling. A broad spectrum analysis of distribution of all the Rab family members across 52 breast cancer cell lines reflected a dichotomous distribution of Rab25, with higher mRNA and protein expression in ER+ cell lines. Rab 25 is of immense interest to us because of its unique substitution of leucine for glutamine at position 71 which typically decreases the intrinsic GTPase activity, resulting in a dominant, constitutively active protein, akin to a transforming RAS mutation. Here we evaluate the role of Rab25 as a valuable predictive and prognostic marker for ER+ breast cancers, with critical implication in disease management and predicting response to therapy. Using high throughput data analysis from publicly available mRNA expression datasets, we report that Rab25 and its effector Rab coupling protein (RCP) are frequently coamplified and coordinately elevated in ER +ve breast cancers. Specifically in the luminal B subtypes, they are significantly associated with a markedly worsened outcome. Compatible with Rab25 and RCP being drivers of the amplicons, their respective mRNA levels are highly correlated across multiple tumor sets, most prominently in luminal B tumors (Soritiou p<0.0015; Pawitan p<0.01). Patients with low RNA levels of both Rab25 and RCP have a very good outcome, those with high level of either Rab25 or RCP have an intermediate outcome and patients with coordinate elevation of Rab25 and RCP mRNA have a bad outcome. Additionally, using our Reverse Phase Protein Array (RPPA) technology, we found that at protein level Rab25 and RCP correlate with poorer outcome in luminal B tumors. In vitro, using MCF7 cell line, which is a prototype of luminal B subtype, we observed an altered EGFR stability and signaling in Rab25 overexpressing stable clones which could be rescued with shRNA knock-down of Rab25, confirming specificity of Rab25 action. In MCF7 cells overexpressing Rab25, we found higher proliferation index as well as increased cell migration and invasion potentials. When injected as xenografts in mice, Rab25 overexpressing cells formed larger tumors than their wildtype controls, strongly suggesting a role for Rab25 in promoting tumor progression in luminal B tumors. The proposed studies will have a marked impact on our understanding of the mechanisms by which Rab25 and RCP contribute to breast cancer aggressiveness, and also of the therapeutic and clinical potential of Rab25 and the Rab25: RCP complex as a target and marker of the behavior of breast tumors.

Atypical PKC Phosphorylates PAR-1 Kinases to Regulate Localization and Activity

The establishment and maintenance of cellular polarity are essential biological processes that must be maintained throughout the lifetime of eukaryotic organisms. The Par-1 protein kinases are key polarity determinants that have been conserved throughout evolution. Par-1 directs anterior-posterior asymmetry in the one-cell C. elegans embryo and the Drosophila oocyte [1]. In mammalian cells, Par-1 may regulate epithelial cell polarity [2]. Relevant substrates of Par-1 in these pathways are just being identified, but it is not yet known how Par-1 itself is regulated. Here, we demonstrate that human Par-1b (hPar-1b) interacts with and is negatively regulated by atypical PKC. hPar-1b is phosphorylated by aPKC on threonine 595, a residue conserved in Par-1 orthologs in mammals, worms, and flies. The equivalent site in hPar-1a, T564, is phosphorylated in vivo and by aPKC in vitro. Importantly, phosphorylation of hPar-1b on T595 negatively regulates the kinase activity and plasma membrane localization of hPar-1b in vivo. This study establishes a novel functional link between two central determinants of cellular polarity, aPKC and Par-1, and suggests a model by which aPKC may regulate Par-1 in polarized cells.

Molecular hijacking and intracellular chaos

Molecular hijacking and intracellular chaos

Visualization of Membrane Sorting and Fusion in Living Cells using Total Internal Reflection (TIR) and Multicolor Video Microscopy

Post-Golgi sorting of different classes of newly synthesized proteins and lipids is central to the generation and maintenance of cellular polarity. to directly visualize the dynamics and location of apical/basolateral sorting and trafficking we used fast time-lapse multicolor video microscopy in living cells. Specifically, green fluorescent protein color variants (cyan, CFP; yellow, YFP) of apical cargo (GPI-anchored) and basolateral cargo (vesicular stomatitis virus glycoprotein, VSVG) were generated; see FIG 1. Fast dual color fluorescence video microscopy allowed visualization with high temporal and spatial resolution. Our studies revealed that apical and basolateral cargo progressively segregated into large domains in Golgi/TGN structures, excluded resident proteins, and exited in separate transport containers. These carries remained distinct and did not merge with endocytic structures en route to the plasma membrane. Interestingly, our data suggest that the primary sorting occurs by lateral segregation in the Golgi, prior to budding (FIG 2). Further characterization of morphological differences of apical versus basolateral transport carriers was achieved using a specialized microscopy technique called total internal reflection (TIR) microscopy. with this approach only the bottom of the cell (<100 nm) was illuminated by an exponentially decaying evanescent “wave” of light. A series of images, taken at ∼1 second intervals, shows a bright “flash” of fluorescence when the vesicle fuse with the plasma membrane and the fluorophore diffuses into the plasma membrane (FIG 3).

The cell polarity protein ASIP/PAR-3 directly associates with junctional adhesion molecule (JAM).

The establishment and maintenance of cellular polarity are critical for the development of multicellular organisms. PAR (partitioning-defective) proteins were identified in Caenorhabditis elegans as determinants of asymmetric cell division and polarized cell growth. Recently, vertebrate orthologues of two of these proteins, ASIP/PAR-3 and PAR-6, were found to form a signalling complex with the small GTPases Cdc42/Rac1 and with atypical protein kinase C (PKC). Here we show that ASIP/PAR-3 associates with the tight-junction-associated protein junctional adhesion molecule (JAM) in vitro and in vivo. No binding was observed with claudin-1, -4 or -5. In fibroblasts and CHO cells overexpressing JAM, endogenous ASIP is recruited to JAM at sites of cell-cell contact. Over expression of truncated JAM lacking the extracellular part disrupts ASIP/PAR-3 localization at intercellular junctions and delays ASIP/PAR-3 recruitment to newly formed cell junctions. During junction formation, JAM appears early in primordial forms of junctions. Our data suggest that the ASIP/PAR-3-aPKC complex is tethered to tight junctions via its association with JAM, indicating a potential role for JAM in the generation of cell polarity in epithelial cells.

A cell culture medium that supports the differentiation of human retinal pigment epithelium into functionally polarized monolayers. Hu J, Bok D. Mol Vis 2001;7:14–19.

In the eye, the retinal pigment epithelium (RPE) is polarized, with an apex and base. Chee’s Essential Medium (CEM) has supported RPE cells in tissue culture, with maintenance of cellular polarity. Since CEM is no longer commercially available, the authors developed CEM replacement medium and compared tissue culture RPE differentiation and polarity with other media, including DMEMF/12 and DMEM with low or high glucose. The RPE grown in the various tissue culture media were assayed for polarity with Na/K ATPase activity with immunofluorescent antibodies and transepithelial resistance (TER). Bestrophin protein expression was detected as an index of differentiation. The authors found better apical expression of Na/K ATPase RPE grown in the CEM replacement and DMEM high glucose media versus RPE grown in the other media. The CEM replacement media resulted in superior TER of the RPE. Bestrophin was better expressed by RPE grown in the CEM replacement media than by RPE grown in the other media. These results suggest that human RPE cultured in CEM replacement media resulted in functional polarity appropriate for modeling of RPE in vivo function.—Hans E. Grossniklaus

Dynamics of Tubulovesicular Recycling Endosomes in Hippocampal Neurons

Neurons are polarized cells, the activity of which relies on the morphological and functional differences between their axonal and somatodendritic domains. One mechanism for establishing and maintaining neuronal polarity is via the selective targeting of proteins to these domains. The endocytic pathway plays a major role in the generation and maintenance of cellular polarity by selectively sorting and recycling endocytosed plasma membrane proteins. In this study we first show that endogenous syntaxin 13 localizes to tubulovesicular organelles that are present in the somatodendritic and axonal domains of neurons. These organelles contain and actively recycle transferrin receptor and are sensitive to brefeldin A, suggesting that they are analogous to the tubulovesicular recycling endosomes in non-neuronal cells. We next use a syntaxin 13-GFP fusion protein transiently expressed in hippocampal neurons, together with time-lapse microscopy, to study the dynamics of the endosomal system in neurons. The analysis revealed the presence of two distinct classes of syntaxin 13-labeled endosomes: round-oval stationary organelles and highly mobile tubulovesicular structures. The dynamic population of tubulovesicular endosomes travels in both directions along microtubules in dendrites and axons. The mobile organelles appear to fuse with and bud from the stationary endosomes, possibly as a means of delivering and picking up their cargo.

The Aspergillus nidulans sepA gene encodes an FH1/2 protein involved in cytokinesis and the maintenance of cellular polarity.

Cytokinesis (septation) in the fungus Aspergillus nidulans occurs through the formation of a transient actin ring at the incipient division site. Temperature-sensitive mutations in the sepA gene prevent septation and cause defects in the maintenance of cellular polarity, without affecting growth and nuclear division. The sepA gene encodes a member of the growing family of FH1/2 proteins, which appear to have roles in morphogenesis and cytokinesis in organisms such as yeast and Drosophila. Results from temperature shift and immunofluorescence microscopy experiments strongly suggest that sepA function requires a preceding mitosis and that sepA acts prior to actin ring formation. Deletion mutants of sepA exhibit temperature-sensitive growth and severe delays in septation at the permissive temperature, indicating that expression of another gene may compensate for the loss of sepA. Conidiophores formed by sepA mutants exhibit abnormal branching of the stalk and vesicle. These results suggest that sepA interacts with the actin cytoskeleton to promote formation of the actin ring during cytokinesis and that sepA is also required for maintenance of cellular polarity during hyphal growth and asexual morphogenesis.

Differential targeting of facilitative glucose transporters in polarized epithelial cells.

We have examined the intracellular localization of five facilitative glucose transporter proteins, one endogenous (GLUT-1) and four exogenous (GLUT-2, -3, -4, and -5), in polarized epithelial cells. GLUT-2, -3, -4, and -5 were stably transfected into Madin-Darby canine kidney (MDCK) cells, and peptide-specific antibodies were used to establish their distribution by immunofluorescence and immunoelectron-microscopic techniques. GLUT-1 and -2 were predominantly targeted to the basolateral domain of the cell, whereas GLUT-3 and -5 were targeted to the apical plasma membrane. The insulin-regulatable glucose transporter GLUT-4 was found in intracellular tubulovesicular structures beneath the surface of the cell. Vectorial 2-deoxy-D-glucose uptake measurements revealed that approximately 95% of glucose entry into wild-type MDCK cells occurs via the basolateral membranes. In GLUT-3-transfected cells, however, apical glucose uptake increased to approximately 55%; this was not observed in cells expressing the other GLUT isoforms. The discrete and differential intracellular localizations of the various GLUTs, in addition to the high level of sequence homology and predicted secondary structure similarity, render the GLUT family ideal for the study of intrinsic targeting motifs involved in the establishment and maintenance of cellular polarity.