Hierarchy of sorting signals in chimeras of intestinal lactase-phlorizin hydrolase and the influenza virus hemagglutinin

Lactase-phlorizin hydrolase (LPH) is an apical protein in intestinal cells. The location of sorting signals in LPH was investigated by preparing a series of mutants that lacked the LPH cytoplasmic domain or had the cytoplasmic domain of LPH replaced by sequences that comprised basolateral targeting signals and overlapping internalization signals of various potency. These signals are mutants of the cytoplasmic domain of the influenza hemagglutinin (HA), which have been shown to be dominant in targeting HA to the basolateral membrane. The LPH-HA chimeras were expressed in Madin-Darby canine kidney (MDCK) and colon carcinoma (Caco-2) cells, and their transport to the cell surface was analyzed. All of the LPH mutants were targeted correctly to the apical membrane. Furthermore, the LPH-HA chimeras were internalized, indicating that the HA tails were available to interact with the cytoplasmic components of clathrin-coated pits. The introduction of a strong basolateral sorting signal into LPH was not sufficient to override the strong apical signals of the LPH external domain or transmembrane domains. These results show that basolateral sorting signals are not always dominant over apical sorting signals in proteins that contain each and suggest that sorting of basolateral from apical proteins occurs within a common compartment where competition for sorting signals can occur.     

N-聚糖和O-聚糖在极性化细胞蛋白质分拣中的作用

极性化上皮细胞的质膜因其所含蛋白质、脂质等组分不同,可以分为细胞膜顶端和细胞膜基底侧端两个区域,而新合成的蛋白质向这两个区域的有效分拣是上皮细胞维持其自身极性及正常功能所必需的。细胞膜基底侧端蛋白质的分拣主要由位于该蛋白质胞质区的信号肽所介导,关于这方面的研究是比较深入的;而细胞膜顶端蛋白质的分拣机制目前尚未阐明,因而显得比较复杂。近年来,糖类分子作为生物体内细胞识别和调控过程的信息分子日益受到关注,人们通过干扰聚糖合成、基因突变以及构建糖基化缺陷细胞株等实验方法,逐渐地认识到糖类分子在极性化上皮细胞的蛋白质分拣调节中起重要作用。由于糖分子本身结构非常复杂,而且目前缺乏研究糖类分子的有效手段,使得糖生物学的研究远远落后于蛋白质和核酸的研究。从而导致探讨糖类分子在蛋白质分拣过程的具体机制相对来说比较困难。本综述拟简要概括糖类分子中N-聚糖和O-聚糖在极性化上皮细胞的蛋白质分拣过程中的作用,以及两种聚糖在此过程中行使分拣信号功能的可能机制。     

Basolateral sorting signals differ in their ability to redirect apical proteins to the basolateral cell surface

Polarized sorting of membrane proteins in epithelial cells is mediated by cytoplasmic basolateral signals or by apical signals in the transmembrane or exoplasmic domains. Basolateral signals were generally found to be dominant over apical determinants. We have generated chimeric proteins with the cytoplasmic domain of either the asialoglycoprotein receptor H1 or the transferrin receptor, two basolateral proteins, fused to the transmembrane and exoplasmic segments of aminopeptidase N, an apical protein, and analyzed them in Madin-Darby canine kidney cells. Whereas both cytoplasmic sequences induced endocytosis of the chimeras, only that of the transferrin receptor mediated basolateral expression in steady state. The H1 fusion protein, although still largely sorted to the basolateral side in biosynthetic surface transport, was subsequently resorted to the apical cell surface. We tested whether the difference in sorting between trimeric wild-type H1 and the dimeric aminopeptidase chimera was caused by the number of sorting signals presented in the oligomers. Consistent with this hypothesis, the H1 signal was fully functional in a tetrameric fusion protein with the transmembrane and exoplasmic domains of influenza neuraminidase. The results suggest that basolateral signals per se need not be dominant over apical determinants for steady-state polarity and emphasize an important contribution of the valence of signals in polarized sorting.     

Identification of a Cytoplasmic Signal for Apical Transcytosis

Polarized epithelial cells contain apical and basolateral surfaces with distinct protein compositions. To establish and maintain this asymmetry, newly made plasma membrane proteins are sorted in the trans Golgi network for delivery to apical or basolateral surfaces. Signals for basolateral sorting are generally located in the cytoplasmic domain of the protein, whereas signals for apical sorting can be in any part of the protein and can depend on N-linked glycosylation of the protein. Signals for constitutive transcytosis to the apical surface have not been reported. In this study, we used the polymeric immunoglobulin receptor (pIgR), which is biosynthetically delivered to the basolateral surface. There the pIgR can bind a ligand and, with or without bound ligand, the pIgR can then be transcytosed to the apical surface. We found that the glycosylation of the pIgR did not affect the biosynthetic transport of the pIgR. However, glycosylation had an effect on pIgR apical transcytosis. Importantly, analysis of the cytoplasmic tail of the pIgR suggested that a short peptide segment was sufficient to transcytose the pIgR or a neutral reporter from the basolateral to the apical surface. This apical transcytosis sorting signal was not involved in polarized biosynthetic traffic of the pIgR.     

The Cytoplasmic Tail of Rhodopsin Acts as a Novel Apical Sorting Signal in Polarized MDCK Cells

All basolateral sorting signals described to date reside in the cytoplasmic domain of proteins, whereas apical targeting motifs have been found to be lumenal. In this report, we demonstrate that wild-type rhodopsin is targeted to the apical plasma membrane via the TGN upon expression in polarized epithelial MDCK cells. Truncated rhodopsin with a deletion of 32 COOH-terminal residues shows a nonpolar steady-state distribution. Addition of the COOH-terminal 39 residues of rhodopsin redirects the basolateral membrane protein CD7 to the apical membrane. Fusion of rhodopsin''s cytoplasmic tail to a cytosolic protein glutathione S-transferase (GST) also targets this fusion protein (GST–Rho39Tr) to the apical membrane. The targeting of GST–Rho39Tr requires both the terminal 39 amino acids and the palmitoylation membrane anchor signal provided by the rhodopsin sequence. The apical transport of GST–Rho39Tr can be reversibly blocked at the Golgi complex by low temperature and can be altered by brefeldin A treatment. This indicates that the membrane-associated GST–Rho39Tr protein may be sorted along a yet unidentified pathway that is similar to the secretory pathway in polarized MDCK cells. We conclude that the COOH-terminal tail of rhodopsin contains a novel cytoplasmic apical sorting determinant. This finding further indicates that cytoplasmic sorting machinery may exist in MDCK cells for some apically targeted proteins, analogous to that described for basolaterally targeted proteins.     

The epithelial-specific adaptor AP1B mediates post-endocytic recycling to the basolateral membrane

To perform vectorial secretory and transport functions that are critical for the survival of the organism, epithelial cells sort plasma membrane proteins into polarized apical and basolateral domains. Sorting occurs post-synthetically, in the trans Golgi network (TGN) or after internalization from the cell surface in recycling endosomes, and is mediated by apical and basolateral sorting signals embedded in the protein structure. Basolateral sorting signals include tyrosine motifs in the cytoplasmic domain that are structurally similar to signals involved in receptor internalization by clathrin-coated pits. Recently, an epithelial-specific adaptor protein complex, AP1B, was identified. AP-1B recognizes a subset of basolateral tyrosine motifs through its mu 1B subunit. Here, we characterized the post-synthetic and post-endocytic sorting of the fast recycling low density lipoprotein receptor (LDLR) and transferrin receptor (TfR) in LLC-PK1 cells, which lack mu 1B and mis-sort both receptors to the apical surface. Targeting and recycling assays in LLC-PK1 cells, before and after transfection with mu 1B, and in MDCK cells, which express mu 1B constitutively, suggest that AP1B sorts basolateral proteins post-endocytically.     

Cytoplasmic Signals Mediate Apical Early Endosomal Targeting of Endotubin in MDCK Cells

Endotubin is an integral membrane protein that targets into apical endosomes in polarized epithelial cells. Although the role of cytoplasmic targeting signals as mediators of basolateral targeting and endocytosis is well established, it has been suggested that apical targeting requires either N-glycosylation of the ectoplasmic domains or partitioning of macromolecules into glycolipid-rich rafts. However, we have previously shown that the cytoplasmic portion of endotubin possesses signals that are necessary for its proper sorting into the apical early endosomes. To further define the targeting signals involved in this apically directed event, as well as to determine if the cytoplasmic domain was sufficient to mediate apical endosomal targeting, we generated a panel of endotubin and Tac-antigen chimeras and expressed them in Madin–Darby canine kidney cells. We show that both the apically targeting wild-type endotubin and a basolaterally targeted cytoplasmic domain mutant do not associate with rafts and are TX-100 soluble. The cytoplasmic tail of endotubin is sufficient for apical endosomal targeting, as chimeras with the endotubin cytoplasmic domain and Tac transmembrane and extracellular domains are efficiently targeted to the apical endosomal compartment. Furthermore, we show that overexpression of these chimeras results in their missorting to the basolateral membrane, indicating that the apical sorting process is a saturable event. These results show that cells contain machinery in both the biosynthetic and endosomal compartments that recognize cytoplasmic apical sorting signals.     

The rat liver Na(+)/bile acid cotransporter. Importance of the cytoplasmic tail to function and plasma membrane targeting

To understand the potential functions of the cytoplasmic tail of Na(+)/taurocholate cotransporter (Ntcp) and to determine the basolateral sorting mechanisms for this transporter, green fluorescent protein-fused wild type and mutant rat Ntcps were constructed and the transport properties and cellular localization were assessed in transfected COS 7 and Madin-Darby canine kidney (MDCK) cells. Truncation of the 56-amino acid cytoplasmic tail demonstrates that the cytoplasmic tail of rat Ntcp is involved membrane delivery of this protein in nonpolarized and polarized cells and removal of the tail does not affect the bile acid transport function of Ntcp. Using site-directed mutagenesis, two tyrosine residues, Tyr-321 and Tyr-307, in the cytoplasmic tail of Ntcp have been identified as important for the basolateral sorting of rat Ntcp in transfected MDCK cells. Tyr-321 appears to be the major basolateral-sorting determinant, and Tyr-307 acts as a supporting determinant to ensure delivery of the transporter to the basolateral surface, especially at high levels of protein expression. When the two Tyr-based basolateral sorting motifs have been removed, the N-linked carbohydrate groups direct the tyrosine to alanine mutants to the apical surface of transfected MDCK cells. The major basolateral sorting determinant Tyr-321 is within a novel beta-turn unfavorable tetrapeptide Y(321)KAA, which has not been found in any naturally occurring basolateral sorting motifs. Two-dimensional NMR spectroscopy of a 24-mer peptide corresponding to the sequence from Tyr-307 to Thr-330 on the cytoplasmic tail of Ntcp confirms that both the Tyr-321 and Tyr-307 regions do not adopt any turn structure. Since the major motif YKAA contains a beta-turn unfavorable structure, the Ntcp basolateral sorting may not be related to the clathrin-adaptor complex pathway, as is the case for many basolateral proteins.     

Carbohydrate-mediated Golgi to cell surface transport and apical targeting of membrane proteins.

Polarized expression of most epithelial plasma membrane proteins is achieved by selective transport from the Golgi apparatus or from endosomes to a specific cell surface domain. In Madin-Darby canine kidney (MDCK) cells, basolateral sorting generally depends on distinct cytoplasmic targeting determinants. Inactivation of these signals often resulted in apical expression, suggesting that apical transport of transmembrane proteins occurs either by default or is mediated by widely distributed characteristics of membrane glycoproteins. We tested the hypothesis of N-linked carbohydrates acting as apical targeting signals using three different membrane proteins. The first two are normally not glycosylated and the third one is a glycoprotein. In all three cases, N-linked carbohydrates were clearly able to mediate apical targeting and transport. Cell surface transport of proteins containing cytoplasmic basolateral targeting determinants was not significantly affected by N-linked sugars. In the absence of glycosylation and a basolateral sorting signal, the reporter proteins accumulated in the Golgi complex of MDCK as well as CHO cells, indicating that efficient transport from the Golgi apparatus to the cell surface is signal-mediated in polarized and non-polarized cells.     

Differential localization of lipid phosphate phosphatases 1 and 3 to cell surface subdomains in polarized MDCK cells

Lipid phosphate phosphatases (LPPs) are integral membrane proteins with six transmembrane domains that act as ecto-enzymes dephosphorylating a variety of extracellular lipid phosphates. Using polarized MDCK cells stably expressing human LPP1 and LPP3, we found that LPP1 was located exclusively at the apical surface whereas LPP3 was distributed mostly in the basolateral subdomain. We identified a novel apical sorting signal at the N-terminus of LPP1 composed of F(2)DKTRL(7). In the case of LPP3, a dityrosine motif present in the second cytoplasmic portion was identified as basolateral targeting signal. Our work shows that LPP1 and LPP3 are equipped with distinct sorting signals that cause them to differentially localize to the apical vs. the basolateral subdomain, respectively.     

N-glycans mediate apical recycling of the sialomucin endolyn in polarized MDCK cells

Apical and basolateral proteins are maintained within distinct membrane subdomains in polarized epithelial cells by biosynthetic and postendocytic sorting processes. Sorting of basolateral proteins in these processes has been well studied; however, the sorting signals and mechanisms that direct proteins to the apical surface are less well understood. We previously demonstrated that an N-glycan-dependent sorting signal directs the sialomucin endolyn to the apical surface in polarized Madin-Darby canine kidney cells. Terminal processing of a subset of endolyn's N-glycans is key for polarized biosynthetic delivery to the apical membrane. Endolyn is subsequently internalized, and via a cytoplasmic tyrosine-based sorting motif is targeted to lysosomes from where it constitutively cycles to the cell surface. Here, we examine the polarized sorting of endolyn along the postendocytic pathway in polarized cells. Our results suggest that similar N-glycan sorting determinants are required for apical delivery of endolyn along both the biosynthetic and the postendocytic pathways.     

Organization of vesicular trafficking in epithelia

Experiments using mammalian epithelial cell lines have elucidated biosynthetic and recycling pathways for apical and basolateral plasma-membrane proteins, and have identified components that guide apical and basolateral proteins along these pathways. These components include apical and basolateral sorting signals, adaptors for basolateral signals, and docking and fusion proteins for vesicular trafficking. Recent live-cell-imaging studies provide a real-time view of sorting processes in epithelial cells, including key roles for actin, microtubules and motors in the organization of post-Golgi trafficking.     

Measles virus matrix protein specifies apical virus release and glycoprotein sorting in epithelial cells

In polarized epithelial cells measles virus (MV) is predominantly released at the apical cell surface, irrespective of the sorting of its two envelope glycoproteins F and H. It has been reported previously that the viral matrix (M) protein modulates the fusogenic capacity of the viral envelope glycoproteins. Here, extant MV mutants and chimeras were used to determine the role of M protein in the transport of viral glycoproteins and release of progeny virions in polarized epithelial CaCo2 cells. In the absence of M, envelope glycoproteins are sorted to the basolateral surface, suggesting that they possess intrinsic basolateral sorting signals. However, interactions of M with the glycoprotein cytoplasmic tails allow M-glycoprotein co-segregation to the apical surface, suggesting a vectorial function of M to retarget the glycoproteins for apical virion release. Whereas this may allow virus airway shedding, the intrinsic sorting of the glycoproteins to the basolateral surface may account for systemic host infection by allowing efficient cell-cell fusion.     

Importance of the carboxyl-terminal FTSL motif of membrane cofactor protein for basolateral sorting and endocytosis. Positive and negative modulation by signals inside and outside the cytoplasmic tail.

Membrane cofactor protein (MCP), a widely distributed complement regulatory protein, is expressed on the basolateral surface of polarized epithelial cells, and it is not endocytosed. The carboxyl-terminal tetrapeptide (FTSL) is required for polarized surface expression. The ability of this tetrapeptide to serve as an autonomous sorting signal has been analyzed by adding this sequence motif to the C terminus of an apical membrane protein, the influenza A virus hemagglutinin (HA). The recombinant protein HA-FTSL retained the apical localization of the parental HA protein. Substitution of the complete cytoplasmic tail of MCP for the cytoplasmic tail of HA resulted in the targeting of the chimeric protein (HA/MCP) to the basolateral surface suggesting that the carboxyl-terminal FTSL motif is a weak sorting signal that requires additional targeting information from the membrane-proximal part of the cytoplasmic tail of MCP for redirecting an apical protein to the basolateral membrane domain. In contrast to the native HA, the HA-FTSL protein was subject to endocytosis. The basolateral HA/MCP was also found to be internalized and thus differed from the basolateral MCP. This result suggests that the carboxyl-terminal FTSL motif serves as an internalization signal and that in native MCP sorting information outside the cytoplasmic tail counteracts this endocytosis signal. Substitution of a tyrosine for the phenylalanine dramatically increased the internalization with most of the HA-YTSL protein being present intracellularly. Our results are consistent with the view that the interplay of multiple sorting signals and the modification of a well known targeting signal (YTSL) by amino acid exchange (FTSL) determine the constitutive expression of MCP on the basolateral surface of polarized epithelial cells.     

Rat liver dipeptidylpeptidase IV contains competing apical and basolateral targeting information.

Madin-Darby canine kidney (MDCK) cells deliver endogenous apical and basolateral proteins directly to the appropriate domains. We are investigating the molecular signals on a model plasma membrane hydrolase, dipeptidylpeptidase IV (DPPIV). Most newly synthesized rat liver DPPIV is delivered directly to the apical surface of transfected MDCK cells; however, about 20% is delivered first to the basolateral surface and reaches the apical surface via transcytosis (Casanova, J. E., Mishumi, Y., Ikehara, Y., Hubbard, A. L., and Mostov, K. E. (1991) J. Biol. Chem. 266, 24428-24432). A soluble form of DPPIV (solDPPIV) containing only the lumenal domain of the protein was efficiently transported and secreted by stably transfected MDCK cells. If this domain contains apical sorting information, we would expect 80% of the soluble protein to be secreted apically. Surprisingly, 95% of the secreted solDPPIV was found in the apical medium. The high efficiency of apical secretion suggested that the transmembrane domain and cytoplasmic tail of DPPIV might contain competing basolateral targeting information. To test this hypothesis, we investigated the trafficking of a chimera in which the cytoplasmic tail and transmembrane domains of DPPIV were joined to lysozyme, an exogenous protein which should not contain sorting information. This protein was delivered predominantly to the basolateral surface. Our results suggest that the lumenal domain of DPPIV carries dominant apical sorting information while the transmembrane domain and cytoplasmic tail of the molecule contains competing basolateral sorting information.     

Sorting of plasma membrane proteins in epithelial cells.

Proteins follow two routes to reach the correct surface (apical or basolateral) of a polarized epithelial cell: direct sorting from the trans-Golgi network and transcytosis from early endosomes. Several signals have been identified recently that control these sorting events, namely a glycosyl-phosphatidylinositol anchor for apical targeting, a 14-residue cytoplasmic segment of the polymeric immunoglobulin receptor for basolateral targeting, and phosphorylation of a Ser residue for transcytosis of this receptor. The machinery involved is still poorly understood.     

Requirement for galectin-3 in apical protein sorting

The central aspect of epithelial cells is their polarized structure, characterized by two distinct domains of the plasma membrane, the apical and the basolateral membrane. Apical protein sorting requires various signals and different intracellular routes to the cell surface. The first apical targeting motif identified is the membrane anchoring of a polypeptide by glycosyl-phosphatidyl-inositol (GPI). A second group of apical signals involves N- and O-glycans, which are exposed to the luminal side of the sorting organelle. Sucrase-isomaltase (SI) and lactase-phlorizin hydrolase (LPH), which use separate transport platforms for trafficking, are two model proteins for the study of apical protein sorting. In contrast to LPH, SI associates with sphingolipid/cholesterol-enriched membrane microdomains or "lipid rafts". After exit form the trans-Golgi network (TGN), the two proteins travel in distinct vesicle populations, SAVs (SI-associated vesicles) and LAVs (LPH-associated vesicles) . Here, we report the identification of the lectin galectin-3 delivering non-raft-dependent glycoproteins in the lumen of LAVs in a carbohydrate-dependent manner. Depletion of galectin-3 from MDCK cells results in missorting of non-raft-dependent apical membrane proteins to the basolateral cell pole. This suggests a direct role of galectin-3 in apical sorting as a sorting receptor.     

The basolateral targeting signal of CD147 (EMMPRIN) consists of a single leucine and is not recognized by retinal pigment epithelium

CD147, a type I integral membrane protein of the immunoglobulin superfamily, exhibits reversed polarity in retinal pigment epithelium (RPE). CD147 is apical in RPE in contrast to its basolateral localization in extraocular epithelia. This elicited our interest in understanding the basolateral sorting signals of CD147 in prototypic Madin-Darby canine kidney (MDCK) cells. The cytoplasmic domain of CD147 has basolateral sorting information but is devoid of well-characterized basolateral signals, such as tyrosine and di-leucine motifs. Hence, we carried out systematic site-directed mutagenesis to delineate basolateral targeting information in CD147. Our detailed analysis identified a single leucine (252) as the basolateral targeting motif in the cytoplasmic tail of CD147. Four amino acids (243-246) N-terminal to leucine 252 are also critical basolateral determinants of CD147, because deletion of these amino acids leads to mistargeting of CD147 to the apical membranes. We ruled out the involvement of adaptor complex 1B (AP1B) in the basolateral trafficking of CD147, because LLC-PK1 cells lacking AP1B, target CD147 basolaterally. At variance with MDCK cells, the human RPE cell line ARPE-19 does not distinguish between CD147 (WT) and CD147 with leucine 252 mutated to alanine and targets both proteins apically. Thus, our study identifies an atypical basolateral motif of CD147, which comprises a single leucine and is not recognized by RPE cells. This unusual basolateral sorting signal will be useful in unraveling the specialized sorting machinery of RPE cells.     

Targeting of transmembrane protein shrew-1 to adherens junctions is controlled by cytoplasmic sorting motifs

We recently identified transmembrane protein shrew-1 and showed that it is able to target to adherens junctions in polarized epithelial cells. This suggested shrew-1 possesses specific basolateral sorting motifs, which we analyzed by mutational analysis. Systematic mutation of amino acids in putative sorting signals in the cytoplasmic domain of shrew-1 revealed three tyrosines and a dileucine motif necessary for basolateral sorting. Substitution of these amino acids leads to apical localization of shrew-1. By applying tannic acid to either the apical or basolateral part of polarized epithelial cells, thereby blocking vesicle fusion with the plasma membrane, we obtained evidence that the apically localized mutants were primarily targeted to the basolateral membrane and were then redistributed to the apical domain. Further support for a postendocytic sorting mechanism of shrew-1 was obtained by demonstrating that mu1B, a subunit of the epithelial cell-specific adaptor complex AP-1B, interacts with shrew-1. In conclusion, our data provide evidence for a scenario where shrew-1 is primarily delivered to the basolateral membrane by a so far unknown mechanism. Once there, adaptor protein complex AP-1B is involved in retaining shrew-1 at the basolateral membrane by postendocytic sorting mechanisms.     

Basolateral sorting of LDL receptor in MDCK cells: the cytoplasmic domain contains two tyrosine-dependent targeting determinants.

In MDCK cells, transport of membrane proteins to the basolateral plasma membrane has been shown to require a distinct cytoplasmic domain determinant. Although the determinant is often related to signals used for localization in clathrin-coated pits, inactivation of the coated pit domain in the human LDL receptor did not affect basolateral targeting. By expressing mutant and chimeric LDL receptors, we have now identified two independently acting signals that are individually sufficient for basolateral targeting. The two determinants mediate basolateral sorting with different efficiencies, but both contain tyrosine residues critical for activity. The first determinant was colinear with, but distinct from, the coated pit domain of the receptor. The second was found in the C-terminal region of the cytoplasmic domain of the receptor and, although tyrosine-dependent, did not mediate endocytosis. The results suggest that membrane proteins can have functionally redundant signals for basolateral transport and that a tyrosine-containing motif may be a common feature of multiple intracellular sorting events.