Cloning and sequence analysis of desmosomal glycoproteins 2 and 3 (desmocollins): cadherin-like desmosomal adhesion molecules with heterogeneous cytoplasmic domains

Desmosomal glycoproteins 2 and 3 (dg2 and 3) or desmocollins have been implicated in desmosome adhesion. We have obtained a 5.0-kb-long clone for dg3 from a bovine nasal epidermal lambda gt11 cDNA library. Sequence analysis of this clone reveals an open reading frame of 2,517 bases encoding a polypeptide of 839 amino acids. The sequence consists of a signal peptide of 28 amino acids, a precursor sequence of 104 amino acids, and a mature protein of 707 amino acids. The latter has the characteristics of a transmembrane glycoprotein with an extracellular domain of 550 amino acids and a cytoplasmic domain of 122 amino acids. The sequence of a partial clone from the same library shows that dg2 has an alternative COOH terminus that is extended by 54 amino acids. Genomic DNA sequence data show that this arises by splicing out of a 46-bp exon that encodes the COOH-terminal 11 amino acids of dg3 and contains an in-frame stop codon. The extracellular domain of dg3 shows 39.4% protein sequence identity with bovine N-cadherin and 28.4% identity with the other major desmosomal glycoprotein, dg1, or desmoglein. The cytoplasmic domain of dg3 and the partial cytoplasmic domain of dg2 show 23 and 24% identity with bovine N-cadherin, respectively. The results support our previous model for the transmembrane organization of dg2 and 3 (Parrish, E.P., J.E. Marston, D.L. Mattey, H.R. Measures, R. Venning, and D.R. Garrod. 1990. J. Cell Sci. 96:239-248; Holton, J.L., T.P. Kenny, P.K. Legan, J.E. Collins, J.N. Keen, R. Sharma, and D.R. Garrod. 1990. J. Cell Sci. 97:239-246). They suggest that these glycoproteins are specialized for calcium-dependent adhesion in their extracellular domains and, cytoplasmically, for the molecular interactions involved in desmosome plaque formation. Moreover this represents the first example of alternative splicing within the cadherin family of cell adhesion molecules.     

Multiple domains in endoglucanase B (CenB) from Cellulomonas fimi: functions and relatedness to domains in other polypeptides.

Endoglucanase B (CenB) from the bacterium Cellulomonas fimi is divided into five discrete domains by linker sequences rich in proline and hydroxyamino acids (A. Meinke, C. Braun, N. R. Gilkes, D. G. Kilburn, R. C. Miller, Jr., and R. A. J. Warren, J. Bacteriol. 173:308-314, 1991). The catalytic domain of 608 amino acids is at the N terminus. The sequence of the first 477 amino acids in the catalytic domain is related to the sequences of cellulases in family E, which includes procaryotic and eucaryotic enzymes. The sequence of the last 131 amino acids of the catalytic domain is related to sequences present in a number of cellulases from different families. The catalytic domain alone can bind to cellulose, and this binding is mediated at least in part by the C-terminal 131 amino acids. Deletion of these 131 amino acids reduces but does not eliminate activity. The catalytic domain is followed by three domains which are repeats of a 98-amino-acid sequence. The repeats are approximately 50% identical to two repeats of 95 amino acids in a chitinase from Bacillus circulans which are related to fibronectin type III repeats (T. Watanabe, K. Suzuki, K. Oyanagi, K. Ohnishi, and H. Tanaka, J. Biol. Chem. 265:15659-15665, 1990). The C-terminal domain of 101 amino acids is related to sequences, present in a number of bacterial cellulases and xylanases from different families, which form cellulose-binding domains (CBDs). It functions as a CBD when fused to a heterologous polypeptide. Cells of Escherichia coli expressing the wild-type cenB gene accumulate both native CenB and a stable proteolytic fragment of 41 kDa comprising the three repeats and the C-terminal CBD. The 41-kDa polypeptide binds to cellulose but lacks enzymatic activity.     

Mutational analysis of domain I of Pseudomonas exotoxin. Mutations in domain I of Pseudomonas exotoxin which reduce cell binding and animal toxicity

Pseudomonas exotoxin (PE) is a single polypeptide chain that contains 613 amino acids and is arranged into three structural domains. Domain I is responsible for cell recognition, II for translocation of PE across membranes and III for ADP ribosylation of elongation factor 2. Treatment of PE with reagents that react with lysine residues has been shown to lead to a reduction in cytotoxic activity apparently due to a modification of domain I (Pirker, R., FitzGerald, D. J. P., Hamilton, T. C., Ozols, R. F., Willingham, M. C., and Pastan, S. (1985) Cancer Res. 45, 751-757). To determine which lysine residues are important in cell recognition, all 12 lysines in domain I were converted to glutamates by site-directed mutagenesis. Also, two deletion mutants encompassing almost all of domain I (amino acids 4-252) or most of domain I (amino acids 4-224) were studied. The mutant proteins were produced in Escherichia coli, purified, and tested for their cytotoxic activity against Swiss 3T3 cells and in mice. The data indicate that conversion of lysine 57 to glutamate reduces cytotoxic activity towards 3T3 cells 50-100-fold and in mice about 5-fold. Deletion of amino acids 4-224 causes a similar reduction in toxicity towards cells and mice. Deletion of most of the rest of domain I (amino acids 4-252) causes a further reduction in toxicity toward cells and mice indicating this second region between amino acids 225 and 252 of domain I is also important in the toxicity of PE. Competition assays indicated that the ability of PEGlu57 to bind to 3T3 cells was greatly diminished, accounting for its diminished cytotoxic activity.     

p53 domains: structure, oligomerization, and transformation.

Wild-type p53 forms tetramers and multiples of tetramers. Friedman et al. (P. N. Friedman, X. B. Chen, J. Bargonetti, and C. Prives, Proc. Natl. Acad. Sci. USA 90:3319-3323, 1993) have reported that human p53 behaves as a larger molecule during gel filtration than it does during sucrose gradient sedimentation. These differences argue that wild-type p53 has a nonglobular shape. To identify structural and oligomerization domains in p53, we have investigated the physical properties of purified segments of p53. The central, specific DNA-binding domain within murine amino acids 80 to 320 and human amino acids 83 to 323 behaves predominantly as monomers during analysis by sedimentation, gel filtration, and gel electrophoresis. This consistent behavior argues that the central region of p53 is globular in shape. Under appropriate conditions, however, this segment can form transient oligomers without apparent preference for a single oligomeric structure. This region does not enhance transformation by other oncogenes. The biological implications of transient oligomerization by this central segment, therefore, remain to be demonstrated. Like wild-type p53, the C terminus, consisting of murine amino acids 280 to 390 and human amino acids 283 to 393, behaves anomalously during gel filtration and apparently has a nonglobular shape. Within this region, murine amino acids 315 to 350 and human amino acids 323 to 355 are sufficient for assembly of stable tetramers. The finding that murine amino acids 315 to 360 enhance transformation by other oncogenes strongly supports the role of p53 tetramerization in oncogenesis. Amino acids 330 to 390 of murine p53 and amino acids 340 to 393 of human p53, which have been implicated by Sturzbecher et al. in tetramerization (H.-W. Sturzbecher, R. Brain, C. Addison, K. Rudge, M. Remm, M. Grimaldi, E. Keenan, and J. R. Jenkins, Oncogene 7:1513-1523, 1992), do not form stable tetramers under our conditions. Our findings indicate that p53 has at least two autonomous oligomerization domains: a strong tetramerization domain in its C-terminal region and a weaker oligomerization domain in the central DNA binding region of p53. Together, these domains account for the formation of tetramers and multiples of tetramers by wild-type p53. The tetramerization domain is the major determinant of the dominant negative phenotype leading to transformation by mutant p53s.     

Structure-function relationships in diphtheria toxin channels: I. Determining a minimal channel-forming domain

Diphtheria Toxin (DT) is a 535 amino acid exotoxin, whose active form consists of two polypeptide chains linked by an interchain disulphide bond. DT's N-terminal A fragment kills cells by enzymatically inactivating their protein synthetic machinery; its C terminal B chain is required for the binding of toxin to sensitive cells and for the translocation of the A fragment into the cytosol. This B fragment, consisting of its N-terminal T domain (amino acids 191–386) and its C-terminal R domain (amino acids 387–535) is responsible for the ion-conducting channels formed by DT in lipid bilayers and cellular plasma membranes. To further delineate the channel-forming region of DT, we studied channels formed by deletion mutants of DT in lipid bilayer membranes under several pH conditions. Channels formed by mutants containing only the T domain (i.e., lacking the A fragment and/or the R domain), as well as those formed by mutants replacing the R domain with Interleukin-2 (Il–2), have single channel conductances and selectivities essentially identical to those of channels formed by wild-type DT. Furthermore, deleting the N-terminal 118 amino acids of the T domain also has minimal effect on the single channel conductance and selectivity of the mutant channels. Together, these data identify a 61 amino acid stretch of the T domain, corresponding to the region which includes -helices TH8 and TH9 in the crystal structure of DT, as the channel-forming region of the toxin.This work was supported by NIH grants AI22021, AI22848 (R.J.C.), T32 GM07288 (J.A.M.) and GM29210 (A.F.).     

Recombinant expression of a secreted form of the atrial natriuretic peptide clearance receptor

A general structure for the atrial natriuretic peptide clearance receptor (ANP C-receptor) has been proposed based on hydropathicity analysis of the deduced amino acid sequence of this membrane protein (Fuller, F., Porter, J.G., Arfsten, A., Miller, J., Schilling, J., Scarborough, R.M., Lewicki, J.A., and Schenk, D.B. (1988) J. Biol. Chem. 263, 9395-9401). The ANP C-receptor is believed to possess a large amino-terminal extracellular domain (436 amino acids), a single hydrophobic transmembrane anchor (23 amino acids), and a short cytoplasmic tail (37 amino acids). As a means of testing the structure and proposed cellular orientation of this protein, we have employed the technique of in vitro mutagenesis to prepare a receptor mutant (anc-) lacking the transmembrane and cytoplasmic domains. Expression of this mutant in mammalian cells using a vaccinia virus vector results in secretion of a truncated soluble form of the ANP C-receptor which binds native ANP and synthetic ANP analogs with a specificity similar to that of the native ANP C-receptor. In contrast to the native ANP C-receptor that exists predominantly as a homodimer on the cell surface, the secreted receptor exists as a monomeric species. The results are consistent with the proposed structure of this receptor with the amino-terminal domain containing the ANP-binding site oriented extracellular to the plasma membrane. In addition, these data demonstrate that the receptor does not require association with the plasma membrane or its native dimeric configuration in order to bind ANP ligands with high affinity and specificity.     

Exon/intron structure of the human alpha 3(IV) gene encompassing the Goodpasture antigen (alpha 3(IV)NC1). Identification of a potentially antigenic region at the triple helix/NC1 domain junction.

The Goodpasture antigen has been identified as the non-collagenous (NC1) domain of alpha 3(IV), a novel collagen IV chain (Saus, J., Wieslander, J., Langeveld, J., Quinones, S., and Hudson, B.G. (1988) J. Biol. Chem. 263, 13374-13380). In the present study, the exon/intron structure and sequence for 285 amino acids of human alpha 3(IV), comprising 53 amino acids of the triple-helical domain and the complete NC1 domain (232 amino acids), were determined. Based on the comparison of the amino acid sequences of the alpha 1(IV), alpha 2(IV), alpha 3(IV), and alpha 5(IV) NC1 domains, a phylogenetic tree was constructed which indicates that alpha 2(IV) was the first chain to evolve, followed by alpha 3(IV), and then by alpha 1(IV) and alpha 5(IV). The exon/intron structure of these domains is consistent with this evolution model. In addition, it appears that alpha 3(IV) changed most after diverging from the parental gene. Analysis of its primary structure reveals that, at the junction between the triple-helical and NC1 domains, there exists a previously unrecognized, highly hydrophilic region (GLKGKRGDSGSPATWTTR) which is unique to the human alpha 3(IV) chain, containing a cell adhesion motif (RGD) as an integral part of a sequence (KRGDSGSP) conforming to a number of protein kinase recognition sites. Based on primary structure data, we outline new aspects to be explored concerning the molecular basis of collagen IV function and Goodpasture syndrome.     

Molecular characterization of the CAN1 locus in Saccharomyces cerevisiae. A transmembrane protein without N-terminal hydrophobic signal sequence

The complete DNA sequence of the CAN1 locus of the yeast Saccharomyces cerevisiae is presented. The predicted primary translation product consists of 590 amino acids. From the hydropathic profile of the amino acid sequence (as calculated by the algorithm of Kyte and Doolittle (Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132)), one can divide the protein into two distinct regions. The 93-amino acid long N-terminal domain is extremely hydrophilic and does not exhibit any cleavable signal sequence. The rest of the protein (from amino acids 94 to 590) shows features typical for an integral membrane protein. The proposal for the N terminus of the primary translation product is based on results obtained by S1 mapping, insertion mutagenesis, and gene fusion experiments.     

Analysis of the levels of conservation of the J domain among the various types of DnaJ-like proteins

DnaJ-like proteins are defined by the presence of an approximately 73 amino acid region termed the J domain. This region bears similarity to the initial 73 amino acids of the Escherichia coli protein DnaJ. Although the structures of the J domains of E coli DnaJ and human heat shock protein 40 have been solved using nuclear magnetic resonance, no detailed analysis of the amino acid conservation among the J domains of the various DnaJ-like proteins has yet been attempted. A multiple alignment of 223 J domain sequences was performed, and the levels of amino acid conservation at each position were established. It was found that the levels of sequence conservation were particularly high in 'true' DnaJ homologues (ie, those that share full domain conservation with DnaJ) and decreased substantially in those J domains in DnaJ-like proteins that contained no additional similarity to DnaJ outside their J domain. Residues were also identified that could be important for stabilizing the J domain and for mediating the interaction with heat shock protein 70.     

Influence of phosphorylation by protein kinase A on CFTR at the cell surface and endoplasmic reticulum

Most, if not all, of the catalytic activity of the tandem catalytic domain-containing receptor-like protein tyrosine phosphatases (PTPs) resides in the membrane proximal domains (D1), with little to no activity associated with the membrane distal domains (D2). Two point mutations in the D2 domain of PTPalpha, which restore invariant amino acids found in the KNRY motif and WPD loop of all active D1 domains, synergistically confer D1-equivalent kinetic properties towards the phosphotyrosine analogue pNPP, and activate PTPalpha-D2 catalysed phosphopeptide hydrolysis (Lim et al., J. Biol. Chem. 273 (1998) 28986-28993; Buist et al., Biochemistry 38 (1999) 914-922). As all D2 domains lack these two D1-invariant amino acids, we have investigated whether other D2 domains are activated by such point mutations. Mutant PTPepsilon-D2, closely related to PTPalpha-D2 and belonging to a subgroup of D2 domains with minimal and conservative substitutions of D1-invariant amino acids, exhibits synergistic activation towards pNPP but not towards a phosphopeptide substrate. CD45-D2, belonging to another subgroup of D2 domains with considerable substitutions in D1-invariant amino acids, is not activated by these mutations, even in the context of a third mutation which restores the minimal essential active site sequence C(X(5))R, indicating that additional defects are sufficient to preclude catalysis. The ability of the KNRY and WPD replacements to activate PTPepsilon-D2 and PTPalpha-D2, but not CD45-D2, in conjunction with the extent and nature of their wild-type amino acid substitutions, suggests that these D2 domains are representative of two functionally distinct groups of D2 domain.     

Structure of the lac carrier protein of Escherichia coli

Circular dichroic measurements on the lac carrier protein purified from the cytoplasmic membrane of Escherichia coli indicate that 85 +/- 5% of the amino acid residues comprising this integral membrane protein are arranged in helical secondary structures. Analysis of the sequential hydropathic character of this protein by the method of Kyte and Doolittle (J. Mol. Biol. (1982) 157, 105-132) indicates that the protein is composed of at least 12 hydrophobic segments with a mean length of 24 +/- 4 residues/segment. Approximately 70% of the 417 amino acids in the lac carrier are found in these domains. The hydropathic profile, together with the circular dichroic measurements, suggest that the 12 hydrophobic segments are largely in a helical conformation. If the segments are assumed to be alpha-helical, the mean length of each domain approximates the thickness of the most hydrophobic portion of the lipid bilayer. Based on these considerations, it is proposed that the lac carrier protein consists of at least 12 alpha-helical segments that traverse the membrane in a perpendicular sense, i.e. in a fashion similar to bacteriorhodopsin.     

Identification and functional characterization of protein 4.1R and actin-binding sites in erythrocyte beta spectrin: regulation of the interactions by phosphatidylinositol-4,5-bisphosphate

The ternary complex of spectrin, F-actin, and protein 4.1R defines the erythrocyte membrane skeletal network, which governs the stability and elasticity of the membrane. It has been shown that both 4.1R and actin bind to the N-terminal region (residues 1-301) of the spectrin beta chain, which contains two calponin homology domains, designated CH1 and CH2. Here, we show that 4.1R also binds to the separate CH1 and CH2 domains. Unexpectedly, truncation of the CH2 domain by its 20 amino acids, corresponding to its N-terminal alpha helix, was found to greatly enhance its binding to 4.1R. The intact N terminus and the CH1 but not the CH2 domain bind to F-actin, but again, deletion of the first 20 amino acids of the latter exposes an actin-binding activity. As expected, the polypeptide 1-301 inhibits the binding of spectrin dimer to actin and formation of the spectrin-actin-4.1R ternary complex in vitro. Furthermore, the binding of 4.1R to 1-301 is greatly enhanced by PIP(2), implying the existence of a regulatory switch in the cell.     

Mutagenesis of conserved charged amino acids in SLH domains of Thermoanaerobacterium thermosulfurigenes EM1 affects attachment to cell wall sacculi

SLH domains (for surface layer homology) are involved in the attachment of proteins to bacterial cell walls. The data presented here assign the conserved TRAE motif within SLH domains a key role for the binding. The charged amino acids arginine (R) or/and glutamic acid (E) were replaced via site-directed mutagenesis by different amino acids. Effects were visualized in an in vitro binding assay using native cell wall sacculi of Thermoanaerobacterium thermosulfurigenes EM1 and different variants of an SLH protein which consisted of the triplicate SLH domain of xylanase XynA of this bacterium and which was purified after expression in Escherichia coli. The results indicated (1) that the TRAE motif is critical for the binding function of SLH domains, (2) that a functional TRAE motif is necessary in all three domains, (3) that a least one (preferentially positively) charged amino acid in the TRAE motif is required for the functionality of the SLH domain, and (4) that the position of the negatively and positively charged amino acids is important. The finding that the cell wall of T. thermosulfurigenes EM1 contains pyruvate (4 μg mg⁻¹) is in agreement with the hypothesis that pyruvylated secondary cell wall polymers function as ligand for SLH domains.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

A heparin binding site in antithrombin III. Identification, purification, and amino acid sequence

A heparin-binding peptide within antithrombin III (ATIII) was identified by digestion of ATIII with Staphylococcus aureus V8 protease followed by purification on reverse-phase high pressure liquid chromatography using a C-4 column matrix. The column fractions were assayed for their ability to bind heparin by ligand blotting with 125I-fluoresceinamine-heparin as previously described (Smith, J. W., and Knauer, D. J. (1987) Anal. Biochem. 160, 105-114). This analysis identified at least three fractions with heparin binding ability of which the peptide eluting at 25.4 min gave the strongest signal. Amino acid sequence analysis of this peptide gave a partially split sequence which was consistent with regions encompassing amino acids 89-96 and 114-156. These amino acids are present in a 1:1 molar ratio which is consistent with a disulfide linkage between Cys-95 and Cys-128. High affinity heparin competed more effectively for the binding of 125I-fluoresceinamine-heparin to this peptide than low affinity heparin. Chondroitin sulfate did not block the binding of 125I-fluoresceinamine-heparin to the peptide. These data strongly suggest that the isolated peptide represents a native heparin-binding region within intact ATIII. Computer generation of a plot of running charge density of ATIII confirms that the region encompassing amino acid residues 123-141 has the highest positive charge density within the molecule. A hydropathy plot of ATIII was generated using a method similar to that of Kyte and Doolittle (Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132). This plot indicates that amino acid residues 126-140 are exposed to the exterior surface of the molecule. Based on these data, we suggest that the region corresponding to amino acid residues 114-156 is a likely site for the physiological heparin-binding domain of ATIII. We also conclude that the proposed disulfide bridges within the protein are suspect and should be re-examined (Petersen, T. E., Dudek-Wojiechowska, G., Sottrup-Jensen, L., and Magnussun, S. (1979) in The Physiological Inhibitors of Coagulation and Fibrinolysis (Collen, D., Wiman, B., and Verstaeta, M., eds) pp. 43-54, Elsevier Scientific Publishing Co., Amsterdam).     

A lack of peptide binding and decreased thermostability suggests that the CASKIN2 scaffolding protein SH3 domain may be vestigial

Background

CASKIN2 is a neuronal signaling scaffolding protein comprised of multiple ankyrin repeats, two SAM domains, and one SH3 domain. The CASKIN2 SH3 domain for an NMR structural determination because its peptide-binding cleft appeared to deviate from the repertoire of aromatic enriched amino acids that typically bind polyproline-rich sequences.

Results

The structure demonstrated that two non-canonical basic amino acids (K290/R319) in the binding cleft were accommodated well in the SH3 fold. An K290Y/R319W double mutant restoring the typical aromatic amino acids found in the binding cleft resulted in a 20 °C relative increase in the thermal stability. Considering the reduced stability, we speculated that the CASKIN2 SH3 could be a nonfunctional remnant in this scaffolding protein.

Conclusions

While the NMR structure demonstrates that the CASKIN2 SH3 domain is folded, its cleft has suffered two substitutions that prevent it from binding typical polyproline ligands. This observation led us to additionally survey and describe other SH3 domains in the Protein Data Bank that may have similarly lost their ability to promote protein-protein interactions.

     

Human cellular fibronectin: comparison of the carboxyl-terminal portion with rat identifies primary structural domains separated by hypervariable regions

We report the isolation and characterization of four overlapping cDNA clones coding for human cellular fibronectin which continuously cover more than 3 kilobases in length. The nucleotide sequence of these cDNAs has been determined, thus elucidating the amino acid sequence of the C-terminal 794 residues of human fibronectin, which cover the edge of cellular-, heparin-, and fibrin-binding domains of this protein. Comparisons of the nucleotide sequences and the deduced amino acid sequences with those of rat [Schwarzbauer, J. E., Tamkun, J. W., Lemischka, I. R., & Hynes, R. O. (1983) Cell (Cambridge, Mass.) 35, 421] indicate a high degree of conservation at both nucleotide and amino acid levels. Comparison with previously published data on amino acid sequences of bovine fibronectin made it possible to identify structurally important features of the protein during the evolution of human, calf, and rat. The deduced human amino acid sequences contain five type III and three type I repeats of internal homologies. The interspecies conservation in amino acids is more pronounced in regions containing the internal repeats and within each functional domain. The implications of these interspecies conservation and divergence are discussed.     

Complete cDNA sequence of chicken vigilin, a novel protein with amplified and evolutionary conserved domains.

The complete cDNA (4375 bp), coding for a new protein called vigilin, was isolated from chicken chondrocytes. The cDNA shows an open reading frame of 1270 amino acids which are organized in 14 tandemly repeated homologous domains. Each domain consists of two subdomains, one with a conserved sequence motif of 35 amino acids (subdomain A) and another one with a presumptive alpha-helical structure of 21-33 amino acids (subdomain B). 149 amino acids at the N-terminus and 71 amino acids at the C-terminus of vigilin do not show the characteristic domain structure. No sequence characteristic of a signal peptide has been found, which argues for an intracellular localisation of vigilin. Vigilin is highly expressed in freshly isolated chicken chondrocytes but little in chondrocytes after prolonged time in culture. Vigilin mRNA exists in two size species, 4.4 kb and 6.5 kb in length due to the usage of different polyadenylation sites. Comparison of the vigilin sequence with data bases showed a remarkable similarity to protein HX from Saccharomyces cerevisiae [Delahodde, A., Becam, A. M., Perea, J. & Jacq, C. (1986) Nucleic Acids Res. 14, 9213-9214]. The yeast protein consists of eight homologous domains with 11 conserved amino acid residues within a set of 35 amino acids. The N-terminal and C-terminal regions of vigilin and protein HX do not reveal any sequence similarity. These results, together with the demonstration of the characteristic vigilin sequence motif in a human cDNA clone, suggest that the repeats represent evolutionary conserved autonomous domains within a family of proteins found in yeast, chicken and man.     

Beta 1,4-galactosyltransferase: a short NH2-terminal fragment that includes the cytoplasmic and transmembrane domain is sufficient for Golgi retention.

Beta 1,4-galactosyltransferase (beta 1,4-GT) is a Golgi-resident, type II membrane-bound glycoprotein that functions in the coordinate biosynthesis of complex oligosaccharides. Additionally, beta 1,4-GT has been localized to the cell surface of a variety of cell types and tissues where it is proposed to function in intercellular recognition and/or adhesion. Thus beta 1,4-GT is an appropriate molecule to be used in analyzing the molecular basis for retention of a membrane-bound enzyme in the Golgi complex and its subsequent or alternative transport to the cell surface. Previously we have shown that the gene for bovine and murine beta 1,4-GT is unusual in that it specifies a short (SGT) and long (LGT) form of the enzyme (Russo, R. N., Shaper, N. L., and Shaper, J. H. (1990) J. Biol. Chem. 265, 3324-3331). The only difference between the two related forms is in the primary structure of the cytoplasmic domains, where LGT has an NH2-terminal extension of 13 amino acids. In this study, we have tested the hypothesis that LGT and SGT are differentially retained in the Golgi or directed to the cell surface. LGT, SGT or chimeric proteins, containing the NH2-terminal cytoplasmic and transmembrane domain of SGT and LGT fused to the cytoplasmic protein pyruvate kinase, were each stably expressed in Chinese hamster ovary cells. Proteins expressed from each construct were localized by immunofluorescence staining exclusively to a perinuclear region, identified as the Golgi by co-localization with wheat germ agglutinin. Furthermore, the subcellular distribution of both SGT and LGT was restricted to the trans-Golgi compartment as assessed by EM immunoelectron microscopy. These data suggest that both forms of beta 1,4-GT are resident trans-Golgi proteins and that an NH2-terminal segment containing the cytoplasmic and transmembrane domains of SGT (39 amino acids) or LGT (52 amino acids) is sufficient for Golgi retention.