The transmembrane domain of murine alpha-mannosidase IB is a major determinant of Golgi localization

Murine alpha1,2-mannosidase IB is a type II transmembrane protein localized to the Golgi apparatus where it is involved in the biogenesis of complex and hybrid N-glycans. This enzyme consists of a cytoplasmic tail, a transmembrane domain followed by a "stem" region and a large C-terminal catalytic domain. To analyze the determinants of targeting, we constructed various deletion mutants of murine alpha1,2-mannosidase IB as well as alpha1,2-mannosidase IB/yeast alpha1,2-mannosidase and alpha1,2-mannosidase IB/GFP chimeras and localized these proteins by fluorescence microscopy, when expressed transiently in COS7 cells. Replacing the catalytic domain of alpha1,2-mannosidase IB with that of the homologous yeast alpha1,2-mannosidase and deleting the "stem" region in this chimera had no effect on Golgi targeting, but caused increased cell surface localization. The N-terminal tagged protein lacking a catalytic domain was also localized to the Golgi. In the latter case, when the stem region was partially or completely removed, the protein was found in both the ER and the Golgi. A chimera consisting of the alpha1,2-mannosidase IB N-terminal region (cytoplasmic and transmembrane domains plus 10 amino acids of the "stem" region) and GFP was localized mainly to the Golgi. Deletion of 30 out of 35 amino acids in the cytoplasmic tail had no effect on Golgi localization. A GFP chimera lacking the entire cytoplasmic tail was found in both the ER and the Golgi. These results indicate that the transmembrane domain of alpha1,2-mannosidase IB is a major determinant of Golgi localization.     

Localization and targeting of the Saccharomyces cerevisiae Kre2p/Mnt1p alpha 1,2-mannosyltransferase to a medial-Golgi compartment

The yeast Kre2p/Mnt1p alpha 1,2-mannosyltransferase is a type II membrane protein with a short cytoplasmic amino terminus, a membrane- spanning region, and a large catalytic luminal domain containing one N- glycosylation site. Anti-Kre2p/Mnt1p antibodies identify a 60-kD integral membrane protein that is progressively N-glycosylated in an MNN1-dependent manner. Kre2p/Mnt1p is localized in a Golgi compartment that overlaps with that containing the medial-Golgi mannosyltransferase Mnn1p, and distinct from that including the late Golgi protein Kex1p. To determine which regions of Kre2p/Mnt1p are required for Golgi localization, Kre2p/Mnt1p mutant proteins were assembled by substitution of Kre2p domains with equivalent sequences from the vacuolar proteins DPAP B and Pho8p. Chimeric proteins were tested for correct topology, in vitro and in vivo activity, and were localized intracellularly by indirect immunofluorescence. The results demonstrate that the NH2-terminal cytoplasmic domain is necessary for correct Kre2p Golgi localization whereas, the membrane-spanning and stem domains are dispensable. However, in a test of targeting sufficiency, the presence of the entire Kre2p cytoplasmic tail, plus the transmembrane domain and a 36-amino acid residue luminal stem region was required to localize a Pho8p reporter protein to the yeast Golgi.     

Primary structure of beta-galactoside alpha 2,6-sialyltransferase. Conversion of membrane-bound enzyme to soluble forms by cleavage of the NH2-terminal signal anchor

This report describes the primary structure of a rat liver beta-galactoside alpha 2,6-sialyltransferase (EC 2.4.99.1), a Golgi apparatus enzyme involved in the terminal sialylation of N-linked carbohydrate groups of glycoproteins. The complete amino acid sequence was deduced from the nucleotide sequence of cDNA clones of the enzyme. The primary structure suggests that the topology of the enzyme in the Golgi apparatus consists of a short NH2-terminal cytoplasmic domain, a 17-residue hydrophobic sequence which serves as the membrane anchor and signal sequence, and a large lumenal, catalytic domain. NH2-terminal sequence analysis of a truncated form of the enzyme, obtained by purification from tissue homogenates, reveals that it is missing a 63-residue NH2-terminal peptide which includes the membrane binding domain. These and supporting results show that soluble forms of the sialyltransferase can be generated by proteolytic cleavage between the NH2-terminal signal-anchor and the catalytic domain.     

A Role for the Lumenal Domain in Golgi Localization of the Saccharomyces cerevisiae Guanosine Diphosphatase

Integral membrane proteins (IMPs) contain localization signals necessary for targeting to their resident subcellular compartments. To define signals that mediate localization to the Golgi complex, we have analyzed a resident IMP of the Saccharomyces cerevisiae Golgi complex, guanosine diphosphatase (GDPase). GDPase, which is necessary for Golgi-specific glycosylation reactions, is a type II IMP with a short amino-terminal cytoplasmic domain, a single transmembrane domain (TMD), and a large catalytic lumenal domain. Regions specifying Golgi localization were identified by analyzing recombinant proteins either lacking GDPase domains or containing corresponding domains from type II vacuolar IMPs. Neither deletion nor substitution of the GDPase cytoplasmic domain perturbed Golgi localization. Exchanging the GDPase TMD with vacuolar protein TMDs only marginally affected Golgi localization. Replacement of the lumenal domain resulted in mislocalization of the chimeric protein from the Golgi to the vacuole, but a similar substitution leaving 34 amino acids of the GDPase lumenal domain intact was properly localized. These results identify a major Golgi localization determinant in the membrane-adjacent lumenal region (stem) of GDPase. Although necessary, the stem domain is not sufficient to mediate localization; in addition, a membrane-anchoring domain and either the cytoplasmic or full-length lumenal domain must be present to maintain Golgi residence. The importance of lumenal domain sequences in GDPase Golgi localization and the requirement for multiple hydrophilic protein domains support a model for Golgi localization invoking protein–protein interactions rather than interactions between the TMD and the lipid bilayer.     

Mutations of the Rous sarcoma virus env gene that affect the transport and subcellular location of the glycoprotein products

The envelope glycoproteins of Rous sarcoma virus (RSV), gp85 and gp37, are anchored in the membrane by a 27-amino acid, hydrophobic domain that lies adjacent to a 22-amino acid, cytoplasmic domain at the carboxy terminus of gp37. We have altered these cytoplasmic and transmembrane domains by introducing deletion mutations into the molecularly cloned sequences of a proviral env gene. The effects of the mutations on the transport and subcellular localization of the Rous sarcoma virus glycoproteins were examined in monkey (CV-1) cells using an SV40 expression vector. We found, on the one hand, that replacement of the nonconserved region of the cytoplasmic domain with a longer, unrelated sequence of amino acids (mutant C1) did not alter the rate of transport to the Golgi apparatus nor the appearance of the glycoprotein on the cell surface. Larger deletions, extending into the conserved region of the cytoplasmic domain (mutant C2), resulted in a slower rate of transport to the Golgi apparatus, but did not prevent transport to the cell surface. On the other hand, removal of the entire cytoplasmic and transmembrane domains (mutant C3) did block transport and therefore did not result in secretion of the truncated protein. Our results demonstrate that the C3 polypeptide was not transported to the Golgi apparatus, although it apparently remained in a soluble, nonanchored form in the lumen of the rough endoplasmic reticulum; therefore, it appears that this mutant protein lacks a functional sorting signal. Surprisingly, subcellular localization by internal immunofluorescence revealed that the C3 protein (unlike the wild type) did not accumulate on the nuclear membrane but rather in vesicles distributed throughout the cytoplasm. This observation suggests that the wild-type glycoproteins (and perhaps other membrane-bound or secreted proteins) are specifically transported to the nuclear membrane after their biosynthesis elsewhere in the rough endoplasmic reticulum.     

The signal for Golgi retention of bovine beta 1,4-galactosyltransferase is in the transmembrane domain.

The expression and localization of bovine beta 1,4-galactosyltransferase (Gal T) has been studied in mammalian cells transfected with Gal T cDNA constructs, and the role of the amino-terminal domains of Gal T in Golgi localization examined. Here we demonstrate that the transmembrane (signal/anchor) domain of bovine Gal T contains a positive Golgi retention signal. Bovine Gal T was characterized in transfected cells with anti-bovine Gal T antibodies, affinity-purified from a rabbit antiserum using a bacterial recombinant fusion protein. These affinity-purified antibodies recognized native bovine Gal T and showed minimum cross-reactivity with Gal T from non-bovine sources. Bovine Gal T cDNA was expressed, as active enzyme, transiently in COS-1 cells and stably in murine L cells, and the product was shown to be localized to the Golgi complex by immunofluorescence using the polypeptide-specific antibodies. A low level of surface bovine Gal T was also detected in the transfected L cells by flow cytometry. The removal of 18 of the 24 amino acids from the cytoplasmic domain of bovine Gal T did not alter the Golgi localization of the product transiently expressed in COS-1 cells or stably expressed in L cells. Both the full-length bovine Gal T and the cytoplasmic domain deletion mutant were N-glycosylated in the transfected L cells, indicating both proteins have the correct N(in)/C(out) membrane orientation. Deletion of both the cytoplasmic and signal/anchor domains of bovine Gal T and incorporation of a cleavable signal sequence resulted in a truncated soluble bovine Gal T that was rapidly secreted (within 1 h) from transfected COS-1 cells. Replacement of the signal/anchor domain of bovine Gal T with the signal/anchor domain of the human transferrin receptor resulted in the transport of the hybrid molecule to the cell surface of transfected COS-1 cells. Furthermore, a hybrid construct containing the signal/anchor domain of Gal T with ovalbumin was efficiently retained in the Golgi complex, whereas ovalbumin anchored to the membrane by the transferrin receptor signal/anchor was expressed at the cell surface of transfected COS-1 cells. Overall, these studies show that the hydrophobic, signal/anchor domain of Gal T is both necessary and sufficient for Golgi localization.     

Membrane protein sorting in the yeast secretory pathway: evidence that the vacuole may be the default compartment

The targeting signals of two yeast integral membrane dipeptidyl aminopeptidases (DPAPs), DPAP B and DPAP A, which reside in the vacuole and the Golgi apparatus, respectively, were analyzed. No single domain of DPAP B is required for delivery to the vacuolar membrane, because removal or replacement of either the cytoplasmic, transmembrane, or lumenal domain did not affect the protein's transport to the vacuole. DPAP A was localized by indirect immunofluorescence to non-vacuolar, punctate structures characteristic of the yeast Golgi apparatus. The 118-amino acid cytoplasmic domain of DPAP A is sufficient for retention of the protein in these structures, since replacement of the cytoplasmic domain of DPAP B with that of DPAP A resulted in an immunolocalization pattern indistinguishable from that of wild type DPAP A. Overproduction of DPAP A resulted in its mislocalization to the vacuole, because cells expressing high levels of DPAP A exhibited vacuolar as well as Golgi staining. Deletion of 22 residues of the DPAP A cytoplasmic domain resulted in mislocalization of the mutant protein to the vacuole. Thus, the cytoplasmic domain of DPAP A is both necessary and sufficient for Golgi retention, and removal of the retention signal, or saturation of the retention apparatus by overproducing DPAP A, resulted in transport to the vacuole. Like wild type DPAP B, the delivery of mutant membrane proteins to the vacuole was unaffected in the secretory vesicle-blocked sec1 mutant; thus, transport to the vacuole was not via the plasma membrane followed by endocytosis. These data are consistent with a model in which membrane proteins are delivered to the vacuole along a default pathway.     

Conversion of a Golgi apparatus sialyltransferase to a secretory protein by replacement of the NH2-terminal signal anchor with a signal peptide

The beta-galactoside alpha 2,6 sialyltransferase, an integral membrane protein localized to the trans-region of the Golgi apparatus, has been converted into a catalytically active secreted protein by the replacement of the NH2-terminal signal-anchor domain with the cleavable signal peptide of human gamma-interferon. Pulse-chase analysis of the wild type and recombinant proteins expressed in stably transfected Chinese hamster ovary cells showed that the wild type sialyltransferase (47 kDa) remained cell-associated. In contrast, the signal peptide-sialyltransferase fusion protein yielded an enzymatically active 41-kDa polypeptide which was secreted with a half-time of 2-3 h, consistent with cleavage of the signal peptide. The data indicate that the catalytic domain does not contain sufficient information for retention in the Golgi apparatus and that retention signals are likely to be found in the NH2-terminal 57 amino acids of the wild type enzyme.     

Medial Golgi but not late Golgi glycosyltransferases exist as high molecular weight complexes. Role of luminal domain in complex formation and localization

To investigate the organization of Golgi glycosyltransferases and their mechanism of localization, we have compared the properties of a number of medial and late acting Golgi enzymes. The medial Golgi enzymes, N-acetylglucosaminyltransferase I and II (GnTI and GnTII) required high salt for solubilization and migrated as high molecular weight complexes on sucrose density gradients. In contrast, the late acting Golgi enzymes, beta1,4-galactosyltransferase and alpha1, 2-fucosyltransferase, were readily solubilized in low salt and migrated as monomers/dimers by sucrose density gradient centrifugation. Analysis of membrane-bound GnTI chimeras indicates that the formation of high molecular weight complexes does not require the transmembrane domain and cytoplasmic tail sequences of GnTI. Furthermore, a soluble form of GnTI, containing the stem region and catalytic domain, accumulated in the Golgi prior to secretion, in contrast to beta1,4-galactosyltransferase. Soluble GnTI, which also associated with high molecular weight complexes, was comparable with membrane-bound GnTI in its ability to glycosylate newly synthesized glycoproteins in vivo. Mutation of charged residues within the stem region of GnTI, known to be important for "kin recognition", had no effect on the efficiency of Golgi localization, the inclusion into high molecular weight complexes, nor functional activity in vivo. The differences in behavior between the medial and late acting Golgi enzymes may contribute to their differential localization and their ability to glycosylate efficiently in the correct Golgi subcompartment.     

Membrane protein retention in the yeast Golgi apparatus: dipeptidyl aminopeptidase A is retained by a cytoplasmic signal containing aromatic residues

The mechanism by which yeast dipeptidyl aminopeptidase (DPAP) A, type II integral membrane protein, is retained in the late Golgi apparatus has been investigated. Prior work demonstrated that the 118-amino acid cytoplasmic domain is both necessary and sufficient for Golgi retention and that mutant or overexpressed DPAP A no longer retained in the Golgi was delivered directly to the vacuolar membrane (Roberts, C. J., S. F. Nothwehr, and T. H. Stevens. 1992. J. Cell Biol. 119:69-83). Replacement of the DPAP A transmembrane domain with a synthetic hydrophobic sequence did not affect either Golgi retention of DPAP A or vacuolar delivery of the retention-defective form of DPAP A. These results indicate that the DPAP A transmembrane domain is not involved in either Golgi retention or targeting of this membrane protein. A detailed mutational analysis of the cytoplasmic domain of DPAP A indicated that the most important elements for retention were within the eight residue stretch 85-92. A 10-amino acid region from DPAP A (81- 90) was sufficient for Golgi retention of alkaline phosphatase, a type II vacuolar membrane protein. Detailed mutational analysis within this 10-amino acid sufficient region demonstrated that a Phe-X-Phe-X-Asp motif was absolutely required for efficient retention. The efficiency of Golgi retention via the DPAP A signal could be diminished by overexpression of wild type but not retention-defective versions of Kex2p, another late Golgi membrane protein, suggesting that multiple Golgi membrane proteins may be retained by a common machinery. These results imply a role for a cytoplasmic signal involving aromatic residues in retention of late Golgi membrane proteins in the yeast Saccharomyces cerevisiae.     

N-terminal motifs in some plant disease resistance proteins function in membrane attachment and contribute to disease resistance

To investigate the role of N-terminal domains of plant disease resistance proteins in membrane targeting, the N termini of a number of Arabidopsis and flax disease resistance proteins were fused to green fluorescent protein (GFP) and the fusion proteins localized in planta using confocal microscopy. The N termini of the Arabidopsis RPP1-WsB and RPS5 resistance proteins and the PBS1 protein, which is required for RPS5 resistance, targeted GFP to the plasma membrane, and mutation of predicted myristoylation and potential palmitoylation sites resulted in a shift to nucleocytosolic localization. The N-terminal domain of the membrane-attached Arabidopsis RPS2 resistance protein was targeted incompletely to the plasma membrane. In contrast, the N-terminal domains of the Arabidopsis RPP1-WsA and flax L6 and M resistance proteins, which carry predicted signal anchors, were targeted to the endomembrane system, RPP1-WsA to the endoplasmic reticulum and the Golgi apparatus, L6 to the Golgi apparatus, and M to the tonoplast. Full-length L6 was also targeted to the Golgi apparatus. Site-directed mutagenesis of six nonconserved amino acid residues in the signal anchor domains of L6 and M was used to change the localization of the L6 N-terminal fusion protein to that of M and vice versa, showing that these residues control the targeting specificity of the signal anchor. Replacement of the signal anchor domain of L6 by that of M did not affect L6 protein accumulation or resistance against flax rust expressing AvrL567 but removal of the signal anchor domain reduced L6 protein accumulation and L6 resistance, suggesting that membrane attachment is required to stabilize the L6 protein.     

Multiple signals are required for alpha2,6-sialyltransferase (ST6Gal I) oligomerization and Golgi localization

A single amino acid difference in the catalytic domain of two isoforms of the alpha2,6-sialyltransferase (ST6Gal I) leads to differences in their trafficking, processing, and oligomerization. The STtyr isoform is transiently localized in the Golgi and is ultimately cleaved and secreted, whereas the STcys isoform is stably localized in the Golgi and is not cleaved and secreted. The stable localization of STcys is correlated with its enhanced ability to oligomerize. To test the hypothesis that multiple signals can mediate Golgi localization and further evaluate the role of oligomerization in the localization process, we evaluated the effects of individually and simultaneously altering the cytosolic tail and transmembrane region of the STcys isoform. We found that the localization, processing, and oligomerization of STcys were not substantially changed when either the core amino acids of the cytosolic tail were deleted or the sequence and length of the transmembrane region were altered. In contrast, when these changes were made simultaneously, the STcys isoform was converted into a form that was processed, secreted, and weakly oligomerized like STtyr. We propose that STcys oligomerization is a secondary event resulting from its concentration in the Golgi via mechanisms independently mediated by its cytosolic tail and transmembrane region.     

The cytoplasmic tail of alpha 1,2-fucosyltransferase contains a sequence for golgi localization

The Golgi apparatus has a central role in the glycosylation of proteins and lipids. There is a sequential addition of carbohydrates by glycosyltransferases that are distributed within the Golgi in the order in which the glycosylation occurs. The mechanism of glycosyltransferase retention is considered to involve their transmembrane domains and flanking regions, although we have shown that the cytoplasmic tail of alpha1,2-fucosyltransferase is important for its Golgi localization. Here we show that the removal of the alpha1,2-fucosyltransferase cytoplasmic tail altered its function of fucosylation and its localization site. When the tail was removed, the enzyme moved from the Golgi to the trans Golgi network, suggesting that the transmembrane is responsible for retention and that the cytoplasmic tail is responsible for localization. The cytoplasmic tail of alpha1,2-fucosyltransferase contains 8 amino acids (MWVPSRRH), and mutating these to alanine indicated a role for amino acids 3 to 7 in localization with a particular role of Ser(5). Mutagenesis of Ser(5) to amino acids containing an hydroxyl (Tyr and Thr) demonstrated that the hydroxyl at position 5 is important. Thus, the cytoplasmic tail, and especially a single amino acid, has a predominant role in the localization and thus the function of alpha1,2-fucosyltransferase.     

Differential subcompartmentation of terminal glycosylation in the Golgi apparatus of intestinal absorptive and goblet cells

Two terminal glycosyltransferases, a sialyltransferase and the blood group A alpha 1,3 N-acetylgalactosaminyltransferase, were found to exhibit differential subcompartmentation in the Golgi apparatus of intestinal goblet and absorptive cells. As expected from their role in terminal glycosylation, the two glycosyltransferases and their products, sialic acid residues and blood group A substance, were localized in the trans cisternae of the Golgi apparatus of goblet cells. In contrast, however, they were found throughout the Golgi apparatus stack of adjacent absorptive cells, with the exception of the fenestrated first cis cisterna. The results are in contrast to the general view that enzymes in the glycosylation pathway are arranged in a cis to trans gradient across the Golgi apparatus and that such polarized distributions may instead be cell type-specific.     

The cytoplasmic tail of alpha 1,3-galactosyltransferase inhibits Golgi localization of the full-length enzyme.

It is currently under debate whether the mechanism of Golgi retention of different glycosyltransferases is determined by sequences in the transmembrane, luminal, or cytoplasmic domains or a combination of these domains. We have shown that the cytoplasmic domains of alpha1,3-galactosyltransferase (GT) and alpha1,2-fucosyltransferase (FT) are involved in Golgi localization. Here we show that the cytoplasmic tails of GT and FT are sufficient to confer specific Golgi localization. Further, we show that the expression of only the cytoplasmic tail of GT can lead to displacement or inhibition of binding of the whole transferase and that cells expressing the cytoplasmic tail of GT were not able to express full-length GT or its product, Galalpha1,3Gal. Thus, the presence of the cytoplasmic tail prevented the localization and function of full-length GT, suggesting a possible specific Golgi binding site for GT. The effect was not altered by the inclusion of the transmembrane domain. Although the transmembrane domain may act as an anchor, these data show that, for GT, only the cytoplasmic tail is involved in specific localization to the Golgi.     

Characterization of the Golgi retention motif of Rift Valley fever virus G(N) glycoprotein

As Rift Valley fever (RVF) virus, and probably all members of the family Bunyaviridae, matures in the Golgi apparatus, the targeting of the virus glycoproteins to the Golgi apparatus plays a pivotal role in the virus replication cycle. No consensus Golgi localization motif appears to be shared among the glycoproteins of these viruses. The viruses of the family Bunyaviridae synthesize their glycoproteins, G(N) and G(C), as a polyprotein. The Golgi localization signal of RVF virus has been shown to reside within the G(N) protein by use of a plasmid-based transient expression system to synthesize individual G(N) and G(C) proteins. While the distribution of individually expressed G(N) significantly overlaps with cellular Golgi proteins such as beta-COP and GS-28, G(C) expressed in the absence of G(N) localizes to the endoplasmic reticulum. Further analysis of expressed G(N) truncated proteins and green fluorescent protein/G(N) chimeric proteins demonstrated that the RVF virus Golgi localization signal mapped to a 48-amino-acid region of G(N) encompassing the 20-amino-acid transmembrane domain and the adjacent 28 amino acids of the cytosolic tail.     

Sequences within and adjacent to the transmembrane segment of alpha-2,6-sialyltransferase specify Golgi retention.

The glycosyltransferase alpha-2,6-sialyltransferase (ST) is a Type II membrane protein localized to the Golgi apparatus. The first 44 amino acids of this protein were able to specify Golgi retention of a fused marker protein, lysozyme. This section of ST contains a transmembrane segment which serves as a non-cleaved signal anchor. When lysozyme was fused to an equivalent region of a cell surface protein it now appeared on the cell surface. Analysis of chimeras between the two proteins revealed that the transmembrane segment of ST specifies Golgi retention. Furthermore, altering this segment in full-length ST results in the protein accumulating on the cell surface. However, the retaining effect of the transmembrane domain of ST is augmented by the presence of adjacent lumenal and cytoplasmic sequences from ST. If these sequences are spaced apart by a transmembrane domain of the same length as that of ST they too can specify Golgi retention. Thus retention in the Golgi of ST appears to involve recognition of an extended region of the protein within and on both sides of the bilayer.     

Structure/function of the human Ga1beta1,3-glucuronosyltransferase. Dimerization and functional activity are mediated by two crucial cysteine residues

Galbeta1,3-glucuronosyltransferase (GlcAT-I) that catalyzes the transfer of a glucuronic acid residue onto the trisaccharide primer of the glycosaminoglycan-protein linkage region plays an essential role in the early steps of the biosynthesis of glycosaminoglycans. In order to gain insight into the structure/function of the enzyme, the human recombinant GlcAT-I was successfully expressed in the yeast Pichia pastoris, with an apparent molecular mass of 43 kDa. Analysis of the electrophoretic mobility of the membrane-bound protein in nonreducing and reducing conditions, together with cross-linking studies, indicated that the membrane-bound GlcAT-I formed active disulfide-linked dimers. GlcAT-I expressed without the predicted N-terminal cytoplasmic tail or secreted as a polypeptide lacking the cytoplasmic tail and transmembrane domain was similarly organized as dimers, suggesting that the structural determinants for the dimerization state are localized in the luminal domain of the protein. In addition, the role of Cys(33) and Cys(301) in that process was investigated by site-directed mutagenesis combined with chemical modification of GlcAT-I by N-phenylmaleimide. Replacement of Cys(33) with alanine abolished the formation of dimers with a concomitant decrease in the catalytic efficiency mainly due to a decrease in apparent maximal velocity and in affinity for UDP-glucuronic acid. On the other hand, N-phenylmaleimide treatment or alanine substitution of the Cys(301) residue inactivated the enzyme. Our study demonstrates that GlcAT-I is organized as a homodimer as a result of disulfide bond formation mediated by Cys(33) localized in the stem region, whereas the residue Cys(301) localized in a conserved C-terminal domain is strictly required for the functional integrity of the enzyme.     

Identification and characterization of PKNbeta, a novel isoform of protein kinase PKN: expression and arachidonic acid dependency are different from those of PKNalpha.

The cDNA clone encoding a novel isoform of protein kinase PKN, termed PKNbeta, was isolated from a HeLa cDNA library. PKNbeta had high sequence homology with PKNalpha, originally isolated PKN, especially in the repeats of charged amino acid-rich region with leucine-zipper like sequences (CZ region/HR1), in the carboxyl-terminal catalytic domain, and in approximately 130 amino acid stretch (D region/HR2), located between CZ region/HR1 and the catalytic domain. However, the amino acid sequence of PKNbeta differed from that of PKNalpha in the region immediately amino-terminal to the catalytic domain, which contained two distinct proline-rich sequences consistent with the class II consensus sequence, PXXPXR, for binding to SH3 domain. Distribution of PKNbeta differed from that of PKNalpha in the following two respects: (1) Northern blotting indicated that PKNbeta mRNA could not be detected in human adult tissues, but was expressed abundantly in human cancer cell lines; (2) immunochemical analysis indicated that PKNbeta localized in nucleus and perinuclear Golgi apparatus, and was almost absent in cytoplasmic region in NIH3T3 cells. Recombinant PKNbeta expressed in COS7 cells displayed autophosphorylation and peptide kinase activity, but was found to be significantly less responsive to arachidonic acid than PKNalpha. The identification of this novel isoform underscores the diversity of PKN signaling pathway.