Polarized sorting and trafficking in epithelial cells

The polarized distribution of proteins and lipids at the surface membrane of epithelial cells results in the formation of an apical and a basolateral domain, which are separated by tight junctions. The generation and maintenance of epithelial polarity require elaborate mechanisms that guarantee correct sorting and vectorial delivery of cargo molecules. This dynamic process involves the interaction of sorting signals with sorting machineries and the formation of transport carriers. Here we review the recent advances in the field of polarized sorting in epithelial cells. We especially highlight the role of lipid rafts in apical sorting.     

Basolateral sorting of transforming growth factor-alpha precursor in polarized epithelial cells: characterization of cytoplasmic domain determinants

In polarized Madin-Darby canine kidney (MDCK) cells, newly synthesized transforming growth factor-alpha precursor (proTGFalpha) is directly sorted to the basolateral cell surface where it is sequentially cleaved and released into the basolateral conditioned medium (Dempsey, P.J., Coffey, R.J., J. Biol. Chem. 269 (1994) 16878-16889). In the present study, the role of the proTGFalpha cytoplasmic domain in basolateral sorting has been investigated using deletional and site-directed mutagenesis, as well as chimeric analyses of different TGFalpha constructs stably expressed in MDCK cells. The loss of polarized secretion of a proTGFalpha secretory mutant (TGFsec88) indicated that the proTGFalpha transmembrane and/or cytoplasmic domains contain essential basolateral sorting information. Using reporter chimeras with two apically sorted membrane proteins, p75 neurotrophin growth factor receptor and placental alkaline phosphatase, we show that the proTGFalpha cytoplasmic domain contains dominant basolateral sorting information. Analysis of proTGFalpha cytoplasmic domain truncation and internal deletion mutants, together with site-directed mutagenesis studies within the full-length proTGFalpha cytoplasmic domain, revealed redundant basolateral sorting motifs. Importantly, the C-terminal type I PDZ-binding motif was not required for basolateral sorting as determined by the integrity of basolateral sorting in deletion mutants lacking this motif. ProTGFalpha basolateral sorting may have important consequences for ligand presentation and spatial compartmentalization of epidermal growth factor receptor signaling networks in polarized epithelial cells.     

Sphingomyelin synthase 1-generated sphingomyelin plays an important role in transferrin trafficking and cell proliferation

Transferrin (Tf) endocytosis and recycling are essential for iron uptake and the regulation of cell proliferation. Tf and Tf receptor (TfR) complexes are internalized via clathrin-coated pits composed of a variety of proteins and lipids and pass through early endosomes to recycling endosomes. We investigated the role of sphingomyelin (SM) synthases (SMS1 and SMS2) in clathrin-dependent trafficking of Tf and cell proliferation. We employed SM-deficient lymphoma cells that lacked SMSs and that failed to proliferate in response to Tf. Transfection of SMS1, but not SMS2, enabled these cells to incorporate SM into the plasma membrane, restoring Tf-mediated proliferation. SM-deficient cells showed a significant reduction in clathrin-dependent Tf uptake compared with the parental SM-producing cells. Both SMS1 gene transfection and exogenous short-chain SM treatment increased clathrin-dependent Tf uptake in SM-deficient cells, with the Tf being subsequently sorted to Rab11-positive recycling endosomes. We observed trafficking of the internalized Tf to late/endolysosomal compartments, and this was not dependent on the clathrin pathway in SM-deficient cells. Thus, SMS1-mediated SM synthesis directs Tf-TfR to undergo clathrin-dependent endocytosis and recycling, promoting the proliferation of lymphoma cells.     

Sphingolipid metabolism in neural cells

Sphingolipids were discovered more than a century ago in the brain. Cerebrosides and sphingomyelins were named so because they were first isolated from neural tissue. Although glycosphingolipids and especially those containing sialic acid in their oligosaccharide moiety are particularly abundant in the brain, sphingolipids are ubiquitous cellular membrane components. They form cell- and species-specific profiles at the cell surfaces that characteristically change in development, differentiation, and oncogenic transformation, indicating the significance of these lipid molecules for cell-cell and cell-matrix interactions as well as for cell adhesion, modulation of membrane receptors and signal transduction. This review summarizes sphingolipid metabolism with emphasis on aspects particularly relevant in neural cell types, including neurons, oligodendrocytes and neuroblastoma cells. In addition, the reader is briefly introduced into the methodology of lipid evaluation techniques and also into the putative physiological functions of glycosphingolipids and their metabolites in neural tissue.     

Hrs recognizes a hydrophobic amino acid cluster in cytokine receptors during ubiquitin-independent endosomal sorting

Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is a component of the ESCRT-0 protein complex that captures ubiquitylated cargo proteins and sorts them to the lysosomal pathway. Although Hrs acts as a key transporter for ubiquitin-dependent endosomal sorting, we previously reported that Hrs is also involved in ubiquitin-independent endosomal sorting of interleukin-2 receptor β (IL-2Rβ). Here, we show direct interactions between bacterially expressed Hrs and interleukin-4 receptor α (IL-4Rα), indicating that their binding is not required for ubiquitylation of the receptors, similar to the case for IL-2Rβ. Examinations of the Hrs binding regions of the receptors reveal that a hydrophobic amino acid cluster in both IL-2Rβ and IL-4Rα is essential for the binding. Whereas the wild-type receptors are delivered to LAMP1-positive late endosomes, mutant receptors lacking the hydrophobic amino acid cluster are sorted to lysobisphosphatidic acid-positive late endosomes rather than LAMP1-positive late endosomes. We also show that the degradation of these mutant receptors is attenuated. Accordingly, Hrs functions during ubiquitin-independent endosomal sorting of the receptors by recognizing the hydrophobic amino acid cluster. These findings suggest the existence of a group of cargo proteins that have this hydrophobic amino acid cluster as a ubiquitin-independent sorting signal.     

极化上皮细胞中转运蛋白的分选

上皮组织细胞必须极化其表面区域以执行其转运生理功能。不同膜转运蛋白定位于细胞膜的不同区域,而细胞与细胞之间则须通过紧密连接复合体紧密连接成极化区域,并调节旁细胞途径的通透性。精密的机体要求上皮细胞具备一个筛选装置,用于将新合成的转运蛋白定位于合适的表面区域;转运蛋白本身也必须内含规定其功能位置的分选信号。目前上皮细胞蛋白分选和蛋白质之间相互作用已被逐渐阐明。上皮细胞通过细胞信号转导途径形成极化初始状态,将自己定位于特定位置,调节细胞与细胞之间、细胞与基质之问的相互作用。最近研究发现其信号转导通路的一个成员是一种AMP激活的蛋白激酶（AMP-stimulated protein kinase．AMPK）,它也是细胞能量感受器。     

Endoplasmic reticulum localization of DHHC palmitoyltransferases mediated by lysine-based sorting signals

Intracellular palmitoylation dynamics are regulated by a family of 24 DHHC (aspartate-histidine-histidine-cysteine) palmitoyltransferases, which are localized in a compartment-specific manner. The majority of DHHC proteins localize to endoplasmic reticulum (ER) and Golgi membranes, and a small number target to post-Golgi membranes. To date, there are no reports of the fine mapping of sorting signals in mammalian DHHC proteins; thus, it is unclear how spatial distribution of the DHHC family is achieved. Here, we have identified and characterized lysine-based sorting signals that determine the restricted localization of DHHC4 and DHHC6 to ER membranes. The ER targeting signal in DHHC6 conforms to a KKXX motif, whereas the signal in DHHC4 is a distinct KXX motif. The identified dilysine signals are sufficient to specify ER localization as adding the C-terminal pentapeptide sequences from DHHC4 or DHHC6, which contain these KXX and KKXX motifs, to the C terminus of DHHC3, redistributes this palmitoyltransferase from Golgi to ER membranes. Recent work proposed that palmitoylation of newly synthesized peripheral membrane proteins occurs predominantly at the Golgi. Indeed, previous analyses of the peripheral membrane proteins, SNAP25 and cysteine string protein, are fully consistent with their initial palmitoylation being mediated by Golgi-localized DHHC proteins. Interestingly, ER-localized DHHC3 is able to palmitoylate SNAP25 and cysteine string protein to a similar level as wild-type Golgi-localized DHHC3 in co-expression studies. These results suggest that targeting of intrinsically active DHHC proteins to defined membrane compartments is an important factor contributing to spatially restricted patterns of substrate palmitoylation.     

Clathrin and AP1B: Key roles in basolateral trafficking through trans-endosomal routes

Research following introduction of the MDCK model system to study epithelial polarity (1978) led to an initial paradigm that posited independent roles of the trans Golgi network (TGN) and recycling endosomes (RE) in the generation of, respectively, biosynthetic and recycling routes of plasma membrane (PM) proteins to apical and basolateral PM domains. This model dominated the field for 20 years. However, studies over the past decade and the discovery of the involvement of clathrin and clathrin adaptors in protein trafficking to the basolateral PM has led to a new paradigm. TGN and RE are now believed to cooperate closely in both biosynthetic and recycling trafficking routes. Here, we critically review these recent advances and the questions that remain unanswered.     

pH-dependent cargo sorting from the Golgi

The vacuolar proton-translocating ATPase (V-ATPase) plays a major role in organelle acidification and works together with other ion transporters to maintain pH homeostasis in eukaryotic cells. We analyzed a requirement for V-ATPase activity in protein trafficking in the yeast secretory pathway. Deficiency of V-ATPase activity caused by subunit deletion or glucose deprivation results in missorting of newly synthesized plasma membrane proteins Pma1 and Can1 directly from the Golgi to the vacuole. Vacuolar mislocalization of Pma1 is dependent on Gga adaptors although no Pma1 ubiquitination was detected. Proper cell surface targeting of Pma1 was rescued in V-ATPase-deficient cells by increasing the pH of the medium, suggesting that missorting is the result of aberrant cytosolic pH. In addition to mislocalization of the plasma membrane proteins, Golgi membrane proteins Kex2 and Vrg4 are also missorted to the vacuole upon loss of V-ATPase activity. Because the missorted cargos have distinct trafficking routes, we suggest a pH dependence for multiple cargo sorting events at the Golgi.     

Sphingosine-1-phosphate lyase deficiency produces a pro-inflammatory response while impairing neutrophil trafficking

Sphingosine-1-phosphate (S1P) lyase catalyzes the degradation of S1P, a potent signaling lysosphingolipid. Mice with an inactive S1P lyase gene are impaired in the capacity to degrade S1P, resulting in highly elevated S1P levels. These S1P lyase-deficient mice have low numbers of lymphocytes and high numbers of neutrophils in their blood. We found that the S1P lyase-deficient mice exhibited features of an inflammatory response including elevated levels of pro-inflammatory cytokines and an increased expression of genes in liver associated with an acute-phase response. However, the recruitment of their neutrophils into inflamed tissues was impaired and their neutrophils were defective in migration to chemotactic stimulus. The IL-23/IL-17/granulocyte-colony stimulating factor (G-CSF) cytokine-controlled loop regulating neutrophil homeostasis, which is dependent on neutrophil trafficking to tissues, was disturbed in S1P lyase-deficient mice. Deletion of the S1P4 receptor partially decreased the neutrophilia and inflammation in S1P lyase-deficient mice, implicating S1P receptor signaling in the phenotype. Thus, a genetic block in S1P degradation elicits a pro-inflammatory response but impairs neutrophil migration from blood into tissues.     

Some factors controlling cell polarity in chick retinal pigment epithelial cells in clonal culture

In clonal culture differentiated chick retinal pigmented epithelial (RPE) cells form a monolayer which shows little or no cellular division. The cells usually rest on a basal and reticular lamina and are polarized with their apical surface towards the medium. The apical surface is characterized by apical protrusions, an extensive apical web of microfilaments and junctional complexes which join the apical-lateral borders. A PA/S positive material with a felt-like appearance from the serum component of the medium coats the surfaces of the tissue culture plates. A similar material is found on any membrane filter which has been exposed to medium containing serum. When such a filter brought in contact with the upper surfaces of the RPE cells, the apical surface characteristics are lost, the cells often accumulate Alcian Blue positive material between the cells and the filter and secrete a reticular and a basal lamina, i.e. they establish a second basal surface. Once this has occurred, the cells appear to either detach from the plate and reverse their polarity, or undergo division forming two cell layers. In the latter case new apical surfaces are created between the cell layers but the cells appear to join to form circular structures rather than sheets. These results suggest that contact with this felt-like material initiates formation of a basal surface. They further suggest that where the apical surface has been converted to a basal one the cell attempts to restore the apical surface either by separating from the plate and reversing its polarity or by creating circular structures and developing new apices oriented toward the center of the circle.     

A classical molecular approach to computer simulation of biological sorting

Steinberg's theory of sorting is modified by replacing the free energy minimization principle with dynamical equations of a molecular nature. Correct cellular sorting then follows in all cases where the mixture is in a liquid or a near-liquid state. Computer examples are described and discussed, primarily for two dimensional, but also for three dimensional, interactions.     

Rab13-dependent trafficking of RhoA is required for directional migration and angiogenesis

Angiogenesis requires concomitant remodeling of cell junctions and migration, as exemplified by recent observations of extensive endothelial cell movement along growing blood vessels. We report that a protein complex that regulates cell junctions is required for VEGF-driven directional migration and for angiogenesis in vivo. The complex consists of RhoA and Syx, a RhoA guanine exchange factor cross-linked by the Crumbs polarity protein Mupp1 to angiomotin, a phosphatidylinositol-binding protein. The Syx-associated complex translocates to the leading edge of migrating cells by membrane trafficking that requires the tight junction recycling GTPase Rab13. In turn, Rab13 associates with Grb2, targeting Syx and RhoA to Tyr(1175)-phosphorylated VEGFR2 at the leading edge. Rab13 knockdown in zebrafish impeded sprouting of intersegmental vessels and diminished the directionality of their tip cells. These results indicate that endothelial cell mobility in sprouting vessels is facilitated by shuttling the same protein complex from disassembling junctions to the leading edges of cells.     

Neutrophil elastase sorting involves plasma membrane trafficking requiring the C-terminal propeptide

The primary granules/secretory lysosomes of neutrophils store mature neutrophil elastase (NE) as a luminal protein after proteolytic removal of N-terminal and C-terminal pro-peptides from a proform of NE. The N-terminal pro-peptide prevents premature activation that might be toxic to the cell, but the C-terminal pro-peptide has no defined function. In this study, we investigated the role of the C-terminal pro-peptide in trafficking of NE by expressing, in rat basophilic leukemia (RBL) cells, both wild-type NE and the mutant NE/Delta248-267, which lacks the C-terminal pro-peptide. Both transfected proteins were found to be targeted to secretory lysosomes. In addition, results from antibody ligation and cell-surface biotinylation indicated that proform of NE was targeted to the plasma membrane, and then subjected to endocytosis. The results were supported by the detection of targeting of the proform to the plasma membrane followed by internalization both in RBL cells and normal granulopoietic precursor cells. Targeting of NE to the plasma membrane required the C-terminal pro-peptide as NE/Delta248-267 expressed in RBL cells bypassed plasma membrane trafficking. Our results indicate targeting of a population of NE to the plasma membrane and internalization dependent on the C-terminal NE pro-peptide.     

BACE1 retrograde trafficking is uniquely regulated by the cytoplasmic domain of sortilin

BACE1 (β-site β-amyloid precursor protein (APP)-cleaving enzyme 1) mediates the first proteolytic cleavage of APP, leading to amyloid β-peptide (Aβ) production. It has been reported that BACE1 intracellular trafficking, in particular endosome-to-TGN sorting, is mediated by adaptor complexes, such as retromer and Golgi-localized γ-ear-containing ARF-binding proteins (GGAs). Here we investigated whether sortilin, a Vps10p domain-sorting receptor believed to participate in retromer-mediated transport of select membrane cargoes, contributes to the subcellular trafficking and activity of BACE1. Our initial studies revealed increased levels of sortilin in post-mortem brain tissue of AD patients and that overexpression of sortilin leads to increased BACE1-mediated cleavage of APP in cultured cells. In contrast, RNAi suppression of sortilin results in decreased BACE1-mediated cleavage of APP. We also found that sortilin interacts with BACE1 and that a sortilin construct lacking its cytoplasmic domain, which contains putative retromer sorting motifs, remains bound to BACE1. However, expression of this truncated sortilin redistributes BACE1 from the trans-Golgi network to the endosomes and substantially reduces the retrograde trafficking of BACE1. Site-directed mutagenesis and chimera experiments reveal that the cytoplasmic tail of sortilin, but not those from other VPS10p domain receptors (e.g. SorCs1b and SorLA), plays a unique role in BACE1 trafficking. Our studies suggest a new function for sortilin as a modulator of BACE1 retrograde trafficking and subsequent generation of Aβ.     

The use of yeast two-hybrid screens in studies of protein:protein interactions involved in trafficking

The yeast two-hybrid system has provided a convenient means to both screen for proteins that interact with a protein of interest and to characterise the known interaction between two proteins. Several groups with an interest in the molecular mechanisms that underlie discrete steps along trafficking pathways have exploited the yeast two-hybrid system. Here, we provide a brief background to the technology, attempt to point out some of the pitfalls and benefits of the different systems that can be employed, and mention some of the areas (within the trafficking field) where yeast two-hybrid interaction assays have been particularly informative.     

Plant-specific cation/H+ exchanger 17 and its homologs are endomembrane K+ transporters with roles in protein sorting

The complexity of intracellular compartments in eukaryotic cells evolved to provide distinct environments to regulate processes necessary for cell proliferation and survival. A large family of predicted cation/proton exchangers (CHX), represented by 28 genes in Arabidopsis thaliana, are associated with diverse endomembrane compartments and tissues in plants, although their roles are poorly understood. We expressed a phylogenetically related cluster of CHX genes, encoded by CHX15-CHX20, in yeast and bacterial cells engineered to lack multiple cation-handling mechanisms. Of these, CHX16-CHX20 were implicated in pH homeostasis because their expression rescued the alkaline pH-sensitive growth phenotype of the host yeast strain. A smaller subset, CHX17-CHX19, also conferred tolerance to hygromycin B. Further differences were observed in K(+)- and low pH-dependent growth phenotypes. Although CHX17 did not alter cytoplasmic or vacuolar pH in yeast, CHX20 elicited acidification and alkalization of the cytosol and vacuole, respectively. Using heterologous expression in Escherichia coli strains lacking K(+) uptake systems, we provide evidence for K(+) ((86)Rb) transport mediated by CHX17 and CHX20. Finally, we show that CHX17 and CHX20 affected protein sorting as measured by carboxypeptidase Y secretion in yeast mutants grown at alkaline pH. In plant cells, CHX20-RFP co-localized with an endoplasmic reticulum marker, whereas RFP-tagged CHX17-CHX19 co-localized with prevacuolar compartment and endosome markers. Together, these results suggest that in response to environmental cues, multiple CHX transporters differentially modulate K(+) and pH homeostasis of distinct intracellular compartments, which alter membrane trafficking events likely to be critical for adaptation and survival.