Interaction with mLin-7 alters the targeting of endocytosed transmembrane proteins in mammalian epithelial cells

To investigate the targeting mechanism for proteins bound to the mammalian Lin-7 (mLin-7) PDZ domain, we created receptor protein chimeras composed of the carboxyl-terminal amino acids of LET-23 fused to truncated nerve growth factor receptor/P75. mLin-7 bound to the chimera with a wild-type LET-23 carboxyl-terminal tail (P75t-Let23WT), but not a mutant tail (P75t-Let23MUT). In Madin-Darby canine kidney (MDCK) cells, P75t-Let23WT localized to the basolateral plasma membrane domain, whereas P75t-Let23MUT remained apical. Furthermore, mutant mLin-7 constructs acted as dominant interfering proteins and inhibited the basolateral localization of P75t-Let23WT. The mechanisms for this differential localization were examined further, and, initially, we found that P75t-Let23WT and P75t-Let23MUT were delivered equally to the apical and basolateral plasma membrane domains. Although basolateral retention of P75t-Let23WT, but not P75t-Let23MUT, was observed, the greatest difference in receptor localization was seen in the rapid trafficking of P75t-Let23WT to the basolateral plasma membrane domain after endocytosis, whereas P75t-Let23MUT was degraded in lysosomes, indicating that mLin-7 binding can alter the fate of endocytosed proteins. Altogether, these data support a model for basolateral protein targeting in mammalian epithelial cells dependent on protein-protein interactions with mLin-7, and also suggest a dynamic role for mLin-7 in endosomal sorting.     

Lin-7 targets the Kir 2.3 channel on the basolateral membrane via a L27 domain interaction with CASK

Polarized expression of the Kir 2.3 channel in renal epithelial cells is influenced by the opposing activities of two different PDZ proteins. Mammalian Lin-7 (mLin-7) directly interacts with Kir 2.3 to coordinate basolateral membrane expression, whereas the tax interacting protein 1 (TIP-1), composed of a single PDZ domain, competes for interaction with mLin-7 and drives Kir 2.3 into the endocytic pathway. Here we show that the basolateral targeting function of mLin-7 depends on its L27 domain, which directs interaction with a cognate L27 domain in the basolateral membrane-anchoring protein, calcium/calmodulin-dependent serine protein kinase (CASK). In MDCK cells, the expression of an mLin-7 mutant that lacks the L27 domain displaced Kir 2.3 from the mLin-7/CASK complex and caused the channel to accumulate into large intracellular vesicles that partially colocalized with Rab-11. Conversely, transplantation of the mLin-7 L27 domain to TIP-1 conferred CASK interaction and basolateral targeting of Kir 2.3. Expression of the CASK L27 domain redistributed endogenous mLin-7 to an intracellular compartment and caused Kir 2.3 to accumulate in subapical endosomes. Taken together, these data support a model whereby mLin-7 acts as a PDZ-to-L27 adapter, mediating indirect association of Kir 2.3 with a basolateral membrane scaffold and thereby stabilizing Kir 2.3 at the basolateral membrane.     

A novel and conserved protein-protein interaction domain of mammalian Lin-2/CASK binds and recruits SAP97 to the lateral surface of epithelia

Mammalian Lin-2 (mLin-2)/CASK is a membrane-associated guanylate kinase (MAGUK) and contains multidomain modules that mediate protein-protein interactions important for the establishment and maintenance of neuronal and epithelial cell polarization. The importance of mLin-2/CASK in mammalian development is demonstrated by the fact that mutations in mLin-2/CASK or SAP97, another MAGUK protein, lead to cleft palate in mice. We recently identified a new protein-protein interaction domain, called the L27 domain, which is present twice in mLin-2/CASK. In this report, we further define the binding of the L27C domain of mLin-2/CASK to the L27 domain of mLin-7 and identify the binding partner for L27N of mLin-2/CASK. Biochemical analysis reveals that this L27N domain binds to the N terminus of SAP97, a region that was previously reported to be essential for the lateral membrane recruitment of SAP97 in epithelia. Our colocalization studies, using dominant-negative mLin-2/CASK, show that the association with mLin-2/CASK is crucial for lateral localization of SAP97 in MDCK cells. We also report the identification of a novel isoform of Discs Large, a Drosophila melanogaster orthologue of SAP97, which contains a region highly related to the SAP97 N terminus and which binds Camguk, a Drosophila orthologue of mLin-2/CASK. Our data identify evolutionarily conserved protein-protein interaction domains that link mLin-2/CASK to SAP97 and account for their common phenotype when mutated in mice.     

Identification, sequence, and mapping of the mouse multiple PDZ domain protein gene, Mpdz.

The PDZ domain gained its name from the three proteins that were first seen to have homology by virtue of these domains, the mammalian postsynaptic density protein, PSD-95, the Drosophila discs-large septate junction protein, DLG, and the mammalian epithelial tight-junction protein zona occludens, ZO-1. Over 50 PDZ domain-containing genes have been recognized so far from almost any organism subjected to sequencing, including mammals, nematodes, yeast, plants, and bacteria. The domain consists of an approximately 90-amino-acid-residue unit, which is often repeated in the protein. The majority of residues form a conserved spatial structure while a few amino acids in critical positions confer protein binding specificity. A subgroup of PDZ domains have been shown to recognize a short carboxy-terminal amino acid motif, T/SXV (Ser/Thr-X-Val-COO-), where X is any amino acid. We have identified and completely sequenced a gene, Mpdz, that encodes a mouse protein containing 13 such domains. We have also mapped the gene to a series of overlapping deletions on mouse chromosome 4 and can therefore determine that its function is not essential for embryonic development or neonatal survival.     

Mammalian Crumbs3 is a small transmembrane protein linked to protein associated with Lin-7 (Pals1)

Drosophila Crumbs is a transmembrane protein that plays an important role in epithelial cell polarity and photoreceptor development. Overexpression of Crumbs in Drosophila epithelia expands the apical surface and leads to disruption of cell polarity. Drosophila Crumbs also interacts with two other polarity genes, Stardust and Discs Lost. Recent work has identified a human orthologue of Drosophila Crumbs, known as CRB1, that is mutated in the eye disorders, retinitis pigmentosa and Leber congenital amaurosis. Our work has demonstrated that human CRB1 can form a complex with mammalian orthologues of Stardust and Discs Lost, known as protein associated with Lin-7 (Pals1) and Pals1 associated tight junction (PATJ), respectively. In the current report we have cloned a full length cDNA for a human paralogue of CRB1 called Crumbs3 (CRB3). In contrast to Drosophila Crumbs and CRB1, CRB3 has a very short extracellular domain but like these proteins it has a conserved intracellular domain that allows it to complex with Pals1 and PATJ. Mouse and human CRB3 have identical intracellular domains but divergent extracellular domains except for a conserved N-glycosylation site. CRB3 is localized to the apical surface and tight junctions but the conserved N linked glycosylation site does not appear to be necessary for CRB3 apical targeting. CRB3 is a specialized isoform of the Crumbs protein family that is expressed in epithelia and can tie the apical membrane to the tight junction.     

Mammalian lin-7 stabilizes polarity protein complexes

Mammalian Lin-7 forms a complex with several proteins, including PALS1, that have a role in polarity determination in epithelial cells. In this study we have found that loss of Lin-7 protein from the polarized epithelial cell line Madin-Darby canine kidney II by small hairpin RNA results in defects in tight junction formation as indicated by lowered transepithelial electrical resistance and mislocalization of the tight junction protein ZO-1 after calcium switch. The knock down of Lin-7 also resulted in the loss of expression of several Lin-7 binding partners, including PALS1 and the polarity protein PATJ. The effects of Lin-7 knock down were rescued by the exogenous expression of murine Lin-7 constructs that contained the L27 domain, but not the PDZ domain alone. Furthermore, exogenously expressed PALS1, but not other Lin-7 binding partners, also rescued the effects of Lin-7 knock down, including the restoration of PATJ protein in rescued cell lines. Finally, the effects of Lin-7 knock down appeared to be due to instability of PALS1 protein in the absence of Lin-7, as indicated by an increased rate of PALS1 protein degradation. Taken together, these results indicate that Lin-7 functions in tight junction formation by stabilizing its membrane-associated guanylate kinase binding partner PALS1.     

Computer modelling in combination with in vitro studies reveals similar binding affinities of Drosophila Crumbs for the PDZ domains of Stardust and DmPar-6

Formation of multiprotein complexes is a common theme to pattern a cell, thereby generating spatially and functionally distinct entities at specialised regions. Central components of these complexes are scaffold proteins, which contain several protein-protein interaction domains and provide a platform to recruit a variety of additional components. There is increasing evidence that protein complexes are dynamic structures and that their components can undergo various interactions depending on the cellular context. However, little is known so far about the factors regulating this behaviour. One evolutionarily conserved protein complex, which can be found both in Drosophila and mammalian epithelial cells, is composed of the transmembrane protein Crumbs/Crb3 and the scaffolding proteins Stardust/Pals1 and DPATJ/PATJ, respectively, and localises apically to the zonula adherens. Here we show by in vitro analysis that, similar as in vertebrates, the single PDZ domain of Drosophila DmPar-6 can bind to the four C-terminal amino acids (ERLI) of the transmembrane protein Crumbs. To further evaluate the binding capability of Crumbs to DmPar-6 and the MAGUK protein Stardust, analysis of the PDZ structural database and modelling of the interactions between the C-terminus of Crumbs and the PDZ domains of these two proteins were performed. The results suggest that both PDZ domains bind Crumbs with similar affinities. These data are supported by quantitative yeast two-hybrid interactions. In vivo analysis performed in cell cultures and in the Drosophila embryo show that the cytoplasmic domain of Crumbs can recruit DmPar-6 and DaPKC to the plasma membrane. The data presented here are discussed with respect to possible dynamic interactions between these proteins.     

Structure of an L27 domain heterotrimer from cell polarity complex Patj/Pals1/Mals2 reveals mutually independent L27 domain assembly mode

The assembly of supramolecular complexes in multidomain scaffold proteins is crucial for the control of cell polarity. The scaffold protein of protein associated with Lin-7 1 (Pals1) forms a complex with two other scaffold proteins, Pals-associated tight junction protein (Patj) and mammalian homolog-2 of Lin-7 (Mals2), through its tandem Lin-2 and Lin-7 (L27) domains to regulate apical-basal polarity. Here, we report the crystal structure of a 4-L27 domain-containing heterotrimer derived from the tripartite complex Patj/Pals1/Mals2. The heterotrimer consists of two cognate pairs of heterodimeric L27 domains with similar conformations. Structural analysis and biochemical data further show that the dimers assemble mutually independently. Additionally, such mutually independent assembly of the two heterodimers can be observed in another tripartite complex, Disks large homolog 1 (DLG1)/calcium-calmodulin-dependent serine protein kinase (CASK)/Mals2. Our results reveal a novel mechanism for tandem L27 domain-mediated, supramolecular complex assembly with a mutually independent mode.     

Crumbs3 Is Essential for Proper Epithelial Development and Viability

First identified in Drosophila, the Crumbs (Crb) proteins are important in epithelial polarity, apical membrane formation, and tight junction (TJ) assembly. The conserved Crb intracellular region includes a FERM (band 4.1/ezrin/radixin/moesin) binding domain (FBD) whose mammalian binding partners are not well understood and a PDZ binding motif that interacts with mammalian Pals1 (protein associated with lin seven) (also known as MPP5). Pals1 binds Patj (Pals1-associated tight-junction protein), a multi-PDZ-domain protein that associates with many tight junction proteins. The Crb complex also binds the conserved Par3/Par6/atypical protein kinase C (aPKC) polarity cassette that restricts migration of basolateral proteins through phosphorylation. Here, we describe a Crb3 knockout mouse that demonstrates extensive defects in epithelial morphogenesis. The mice die shortly after birth, with cystic kidneys and proteinaceous debris throughout the lungs. The intestines display villus fusion, apical membrane blebs, and disrupted microvilli. These intestinal defects phenocopy those of Ezrin knockout mice, and we demonstrate an interaction between Crumbs3 and ezrin. Taken together, our data indicate that Crumbs3 is crucial for epithelial morphogenesis and plays a role in linking the apical membrane to the underlying ezrin-containing cytoskeleton.     

Two independent domains of hDlg are sufficient for subcellular targeting: the PDZ1-2 conformational unit and an alternatively spliced domain

hDlg, a human homologue of the Drosophila Dig tumor suppressor, contains two binding sites for protein 4.1, one within a domain containing three PSD-95/Dlg/ZO-1 (PDZ) repeats and another within the alternatively spliced I3 domain. Here, we further define the PDZ- protein 4.1 interaction in vitro and show the functional role of both 4.1 binding sites in situ. A single protease-resistant structure formed by the entirety of both PDZ repeats 1 and 2 (PDZ1-2) contains the protein 4.1-binding site. Both this PDZ1-2 site and the I3 domain associate with a 30-kD NH2-terminal domain of protein 4.1 that is conserved in ezrin/radixin/moesin (ERM) proteins. We show that both protein 4.1 and the ezrin ERM protein interact with the murine form of hDlg in a coprecipitating immune complex. In permeabilized cells and tissues, either the PDZ1-2 domain or the I3 domain alone are sufficient for proper subcellular targeting of exogenous hDlg. In situ, PDZ1-2- mediated targeting involves interactions with both 4.1/ERM proteins and proteins containing the COOH-terminal T/SXV motif. I3-mediated targeting depends exclusively on interactions with 4.1/ERM proteins. Our data elucidates the multivalent nature of membrane-associated guanylate kinase homologue (MAGUK) targeting, thus beginning to define those protein interactions that are critical in MAGUK function.     

Implications of nectin-like molecule-2/IGSF4/RA175/SgIGSF/TSLC1/SynCAM1 in cell-cell adhesion and transmembrane protein localization in epithelial cells

Nectins are Ca2+-independent immunoglobulin-like cell-cell adhesion molecules that play roles in organization of a variety of cell-cell junctions in cooperation with or independently of cadherins. Four nectins have been identified. Five nectin-like molecules, which have domain structures similar to those of nectins, have been identified, and we characterized here nectin-like molecule-2 (Necl-2)/IGSF4/RA175/SgIGSF/TSLC1/SynCAM1. Necl-2 showed Ca2+-independent homophilic cell-cell adhesion activity. It furthermore showed Ca2+-independent heterophilic cell-cell adhesion activity with Necl-1/TSLL1/SynCAM3 and nectin-3. Necl-2 was widely expressed in rat tissues examined. Necl-2 localized at the basolateral plasma membrane in epithelial cells of the mouse gall bladder, but not at specialized cell-cell junctions, such as tight junctions, adherens junctions, and desmosomes. Nectins bind afadin, whereas Necl-2 did not bind afadin but bound Pals2, a membrane-associated guanylate kinase family member known to bind Lin-7, implicated in the proper localization of the Let-23 protein in Caenorhabditis elegans, the homologue of mammalian epidermal growth factor receptor. These results indicate the unique localization of Necl-2 and its possible involvement in localization of a transmembrane protein(s) through Pals2.     

The carboxyl terminus of zona occludens-3 binds and recruits a mammalian homologue of discs lost to tight junctions

Mammalian homologues of the Drosophila polarity proteins Stardust, Discs Lost, and Crumbs have been identified as Pals1, Pals1-associated tight junction protein (PATJ), and human Crumbs homologue 1 (CRB1), respectively. We have previously demonstrated that PATJ, Pals1, and CRB1 can form a tripartite tight junction complex in epithelial cells and that PATJ recruits Pals1 to tight junctions. Here, we observed that the Pals1/PATJ interaction was not crucial for the ultimate targeting of PATJ itself to tight junctions. This prompted us to examine if any of the 10 post-synaptic density-95/Discs Large/zona occludens-1 (PDZ) domains of PATJ could bind to the carboxyl termini of known tight junction constituents. We found that the 6th and 8th PDZ domains of PATJ can interact with the carboxyl termini of zona occludens-3 (ZO-3) and claudin 1, respectively. PATJ missing the 6th PDZ domain was found to mislocalize away from cell contacts. Surprisingly, deleting the 8th PDZ domain had little effect on PATJ localization. Finally, reciprocal co-immunoprecipitation experiments revealed that full-length ZO-3 can associate with PATJ. Hence, the PATJ/ZO-3 interaction is likely important for recruiting PATJ and its associated proteins to tight junctions.     

TIP-1 has PDZ scaffold antagonist activity

PDZ proteins usually contain multiple protein-protein interaction domains and act as molecular scaffolds that are important for the generation and maintenance of cell polarity and cell signaling. Here, we identify and characterize TIP-1 as an atypical PDZ protein that is composed almost entirely of a single PDZ domain and functions as a negative regulator of PDZ-based scaffolding. We found that TIP-1 competes with the basolateral membrane mLin-7/CASK complex for interaction with the potassium channel Kir 2.3 in model renal epithelia. Consequently, polarized plasma membrane expression of Kir 2.3 is disrupted resulting in pronounced endosomal targeting of the channel, similar to the phenotype observed for mutant Kir 2.3 channels lacking the PDZ-binding motif. TIP-1 is ubiquitously expressed, raising the possibility that TIP-1 may play a similar role in regulating the expression of other membrane proteins containing a type I PDZ ligand.     

VAM-1: a new member of the MAGUK family binds to human Veli-1 through a conserved domain

The MAGUKs (membrane-associated guanylate kinase homologues) constitute a family of peripheral membrane proteins that function in tumor suppression and receptor clustering by forming multiprotein complexes containing distinct sets of transmembrane, cytoskeletal, and cytoplasmic signaling proteins. Here, we report the characterization of the human vam-1 gene that encodes a novel member of the p55 subfamily of MAGUKs. The complete cDNA sequence of VAM-1, tissue distribution of its mRNA, genomic structure, chromosomal localization, and Veli-1 binding properties are presented. The vam-1 gene is composed of 12 exons and spans approx. 115 kb. By fluorescence in situ hybridization the vam-1 gene was localized to 7p15-21, a chromosome region frequently disrupted in some human cancers. VAM-1 mRNA was abundant in human testis, brain, and kidney with lower levels detectable in other tissues. The primary structure of VAM-1, predicted from cDNA sequencing, consists of 540 amino acids including a single PDZ domain near the N-terminus, a central SH3 domain, and a C-terminal GUK (guanylate kinase-like) domain. Sequence alignment, heterologous transfection, GST pull-down experiments, and blot overlay assays revealed a conserved domain in VAM-1 that binds to Veli-1, the human homologue of the LIN-7 adaptor protein in Caenorhabditis. LIN-7 is known to play an essential role in the basolateral localization of the LET-23 tyrosine kinase receptor, by linking the receptor to LIN-2 and LIN-10 proteins. Our results therefore suggest that VAM-1 may function by promoting the assembly of a Veli-1 containing protein complex in neuronal as well as epithelial cells.     

Drosophila Lin-7 is a component of the Crumbs complex in epithelia and photoreceptor cells and prevents light-induced retinal degeneration

The Drosophila Crumbs protein complex is required to maintain epithelial cell polarity in the embryo, to ensure proper morphogenesis of photoreceptor cells and to prevent light-dependent retinal degeneration. In Drosophila, the core components of the complex are the transmembrane protein Crumbs, the membrane-associated guanylate kinase (MAGUK) Stardust and the scaffolding protein DPATJ. The composition of the complex and some of its functions are conserved in mammalian epithelial and photoreceptor cells. Here, we report that Drosophila Lin-7, a scaffolding protein with one Lin-2/Lin-7 (L27) domain and one PSD-95/Dlg/ZO-1 (PDZ) domain, is associated with the Crumbs complex in the subapical region of embryonic and follicle epithelia and at the stalk membrane of adult photoreceptor cells. DLin-7 loss-of-function mutants are viable and fertile. While DLin-7 localization depends on Crumbs, neither Crumbs, Stardust nor DPATJ require DLin-7 for proper accumulation in the subapical region. Unlike other components of the Crumbs complex, DLin-7 is also enriched in the first optic ganglion, the lamina, where it co-localizes with Discs large, another member of the MAGUK family. In contrast to crumbs mutant photoreceptor cells, those mutant for DLin-7 do not display any morphogenetic abnormalities. Similar to crumbs mutant eyes, however, DLin-7 mutant photoreceptors undergo progressive, light-dependent degeneration. These results support the previous conclusions that the function of the Crumbs complex in cell survival is independent from its function in photoreceptor morphogenesis.     

Characterization of MPP4, a gene highly expressed in photoreceptor cells,and mutation analysis in retinitis pigmentosa

Membrane-associated guanylate kinase (MAGUK) proteins are cell-cell contact organizing molecules that mediate targeting, clustering and anchoring of proteins at synapses and other cell junctions. MAGUK proteins may contain multiple protein-protein interaction motifs including PDZ, SH3 and guanylate kinase (GuK) domains. In this study, we performed a detailed analysis of the expression pattern of MPP4, a recently described member of the MAGUK protein family. We confirmed that this gene is highly expressed in retina, and demonstrate that it is also present, at lower levels, in brain. We identified a new retina specific isoform encoding a predicted protein lacking 71 amino acids. This protein region contains a newly identified L27 domain, another module playing a role in protein-protein interaction. By RNA in situ hybridization, Mpp4 expression was found to be localized to photoreceptor cells in postnatal retina. The MPP4 gene is localized to chromosome 2, in band 2q31-33, where a locus for autosomal recessive retinitis pigmentosa (RP26) has been mapped. Mutation analysis of the entire open reading frame of the MPP4 gene in a RP26 family revealed no pathologic mutations. In addition, we did not identify mutations in a panel of 300 unrelated patients with retinitis pigmentosa.     

Genetic studies define MAGUK proteins as regulators of epithelial cell polarity

Polarized epithelial cells play critical roles during early embryonic development and organogenesis. Multi-domain scaffolding proteins belonging to the membrane associated guanylate kinase (MAGUK) family are commonly found at the plasma membrane of polarized epithelial cells. Genetic studies in Drosophila melanogaster and Caenorhabditis elegans have revealed that MAGUK proteins regulate various aspects of the polarized epithelial phenotype, including cell junction assembly, targeting of proteins to the plasma membrane and the organisation of polarized signalling complexes. This review will focus on the genetic studies that have contributed to our understanding of the MAGUK family members, Dlg and Lin-2/CASK, in controlling these processes. In addition, our recent genetic analysis of mouse Dlg, in combination with genetic and biochemical studies of Lin-2/CASK by others suggests a model placing Dlg and Lin-2/CASK within the same developmental pathway.     

Molecular characterization of KEX1, a kexin-like protease in mouse Pneumocystis carinii

Expression screening of a Pneumocystis carinii-infected mouse lung cDNA library with specific monoclonal antibodies (mAbs) led to the identification of a P. carinii cDNA with extensive homology to subtilisin-like proteases, particularly fungal kexins and mammalian prohormone convertases. The 3.1 kb cDNA contains a single open reading frame encoding 1011 amino acids. Structural similarities to fungal kexins in the deduced primary amino acid sequence include a putative proenzyme domain delineated by a consensus autocatalytic cleavage site (Arg-Glu-Lys-Arg), conserved Asp, His, Asn and Ser residues in the putative catalytic domain, a hydrophobic transmembrane spanning domain, and a carboxy-terminal cytoplasmic domain with a conserved tyrosine motif thought to be important for localization of the protease in the endoplasmic reticulum and/or Golgi apparatus. Based on these structural similarities and the classification of P. carinii as a fungus, the protease was named KEX1. Southern blotting of mouse P. carinii chromosomes localized kex1 to a single chromosome of approximately 610 kb. Southern blotting of restriction enzyme digests of genomic DNA from P. carinii-infected mouse lung demonstrated that kex1 is a single copy gene. The function of kexins in other fungi suggests that KEX1 may be involved in the post-translational processing and maturation of other P. carinii proteins.     

The GRIP domain - a novel Golgi-targeting domain found in several coiled-coil proteins

Many large coiled-coil proteins are being found associated peripherally with the cytoplasmic face of the organelles of the secretory pathway. Various roles have been proposed for these proteins, including the docking of donor vesicles or organelles to an acceptor organelle prior to fusion, and, in the case of the Golgi apparatus, the stacking of the cisternae [1] [2] [3] [4] [5]. Such critical roles require accurate recruitment to the correct organelle. For the endosomal coiled-coil protein EEA1, targeting requires a carboxy-terminal FYVE domain, which interacts with Rab5 and phosphatidylinositol 3-phosphate (PI(3)P), whereas the Golgi protein GM130 interacts with Golgi membranes via the protein GRASP65 [3] [6] [7]. In this paper, we show that two other mammalian Golgi coiled-coil proteins, golgin-245/p230 and golgin-97, have a conserved domain of about 50 amino acids at their carboxyl termini. This 'GRIP' domain is also found at the carboxyl terminus of several other large coiled-coiled proteins of unknown function, including two human proteins and proteins in the genomes of Caenorhabditis elegans and yeasts. The GRIP domains from several of these proteins, including that from the yeast protein Imh1p, were sufficient to specify Golgi targeting in mammalian cells when fused to green fluorescent protein (GFP). This result suggests that this small domain functions to recruit specific coiled-coil proteins to the Golgi by recognising a determinant that has been well conserved in eukaryotic evolution.