Golgi Localisation Research Articles

Golgi-localized Ring Finger Protein 121 is necessary for MYCN-driven neuroblastoma tumorigenesis

MYCN amplification predicts poor prognosis in childhood neuroblastoma. To identify MYCN oncogenic signal dependencies we performed N-ethyl-N-nitrosourea (ENU) mutagenesis on the germline of neuroblastoma-prone TH-MYCN transgenic mice to generate founders which had lost tumorigenesis. Sequencing of the mutant mouse genomes identified the Ring Finger Protein 121 (RNF121WT) gene mutated to RNFM158R associated with heritable loss of tumorigenicity. While the RNF121WT protein localised predominantly to the cis-Golgi Complex, the RNF121M158R mutation in Helix 4 of its transmembrane domain caused reduced RNF121 protein stability and absent Golgi localisation. RNF121WT expression markedly increased during TH-MYCN tumorigenesis, whereas hemizygous RNF121WT gene deletion reduced TH-MYCN tumorigenicity. The RNF121WT-enhanced growth of MYCN-amplified neuroblastoma cells depended on RNF121WT transmembrane Helix 5. RNF121WT directly bound MYCN protein and enhanced its stability. High RNF121 mRNA expression associated with poor prognosis in human neuroblastoma tissues and another MYC-driven malignancy, laryngeal cancer. RNF121 is thus an essential oncogenic cofactor for MYCN and a target for drug development.

N-glycan Core Tri-fucosylation Requires Golgi α-mannosidase III Activity that Impacts Nematode Growth and Behaviour

N-glycans with complex core chitobiose modifications (CCMs) are observed in various free-living and parasitic nematodes but are absent in mammals. Using Caenorhabditis elegans as a model, we demonstrated that the core N-acetylglucosamine (GlcNAc) residues are modified by three fucosyltransferases, namely FUT-1, FUT-6 and FUT-8. Interestingly, FUT-6 can only fucosylate N-glycans lacking the α1,6-mannose upper arm, indicating that a specific α-mannosidase is required to generate substrates for subsequent FUT-6 activity. By analysing the N-glycomes of aman-3 knockouts using offline HPLC-MALDI-TOF MS/MS, we observed that the absence of aman-3 abolishes α1,3-fucosylation of the distal GlcNAc of N-glycans, which suggests that AMAN-3 is the relevant mannosidase on whose action FUT-6 depends. Enzymatic characterisation of recombinant AMAN-3 and confocal microscopy studies using a knock-in strain (aman-3::eGFP) demonstrated a Golgi localisation. In contrast to the classical Golgi α-mannosidase II (AMAN-2), AMAN-3 displayed a cobalt-dependent α1,6-mannosidase activity towards N-glycans. Using AMAN-3 and other C. elegans glycoenzymes, we were able to mimic nematode N-glycan biosynthesis in vitro by remodelling a fluorescein conjugated-glycan and generate a tri-fucosylated structure. In addition, using a high-content computer-assisted C. elegans analysis platform, we observed that aman-3 deficient worms display significant developmental delays, morphological and behavioural alterations in comparison to the wild type. Our data demonstrated that AMAN-3 is a Golgi α-mannosidase required for core fucosylation of the distal GlcNAc of N-glycans. This enzyme is essential for the formation of the unusual tri-fucosylated CCMs in nematodes, which may play important roles in nematode development and behaviour.

Recruitment of PI4KIIIβ to the Golgi by ACBD3 is dependent on an upstream pathway of a SNARE complex and golgins.

ACBD3 is a protein localised to the Golgi apparatus and recruits other proteins, such as PI4KIIIβ, to the Golgi. However, the mechanism through which ACBD3 itself is recruited to the Golgi is poorly understood. This study demonstrates there are two mechanisms for ACBD3 recruitment to the Golgi. First, we identified that an MWT374-376 motif in the unique region upstream of the GOLD domain in ACBD3 is essential for Golgi localization. Second, we use unbiased proteomics to demonstrate that ACBD3 interacts with SCFD1, a Sec1/Munc-18 (SM) protein, and a SNARE protein, SEC22B. CRISPR-KO of SCFD1 causes ACBD3 to become cytosolic. We also found that ACBD3 is redundantly recruited to the Golgi apparatus by two golgins: golgin-45 and giantin, which bind to ACBD3 through interaction with the MWT374-376 motif. Taken together, our results suggest that ACBD3 is recruited to the Golgi in a two-step sequential process, with the SCFD1-mediated interaction occurring upstream of the interaction with the golgins.

Kinetics of Arf1 inactivation regulates Golgi organisation and function in non-adherent fibroblasts.

Arf1 belongs to the Arf family of small GTPases that localise at the Golgi and plasma membrane. Active Arf1 plays a crucial role in regulating Golgi organisation and function. In mouse fibroblasts, loss of adhesion triggers a consistent drop (∼50%) in Arf1 activation that causes the Golgi to disorganise but not fragment. In suspended cells, the trans-Golgi (GalTase) disperses more prominently than cis-Golgi (Man II), accompanied by increased active Arf1 (detected using GFP-ABD: ARHGAP10 Arf1 binding domain) associated with the cis-Golgi compartment. Re-adhesion restores Arf1 activation at the trans-Golgi as it reorganises. Arf1 activation at the Golgi is regulated by Arf1 Guanine nucleotide exchange factors (GEFs), GBF1, and BIG1/2. In non-adherent fibroblasts, the cis-medial Golgi provides a unique setting to test and understand the role GEF-mediated Arf1 activation has in regulating Golgi organisation. Labelled with Man II-GFP, non-adherent fibroblasts treated with increasing concentrations of Brefeldin-A (BFA) (which inhibits BIG1/2 and GBF1) or Golgicide A (GCA) (which inhibits GBF1 only) comparably decrease active Arf1 levels. They, however, cause a concentration-dependent increase in cis-medial Golgi fragmentation and fusion with the endoplasmic reticulum (ER). Using selected BFA and GCA concentrations, we find a change in the kinetics of Arf1 inactivation could mediate this by regulating cis-medial Golgi localisation of GBF1. On loss of adhesion, a ∼50% drop in Arf1 activation over 120 min causes the Golgi to disorganise. The kinetics of this drop, when altered by BFA or GCA treatment causes a similar decline in Arf1 activation but over 10 min. This causes the Golgi to now fragment which affects cell surface glycosylation and re-adherent cell spreading. Using non-adherent fibroblasts this study reveals the kinetics of Arf1 inactivation, with active Arf1 levels, to be vital for Golgi organisation and function.

Identification and characterisation of a cryptic Golgi complex in Naegleria gruberi.

ABSTRACTAlthough the Golgi complex has a conserved morphology of flattened stacked cisternae in most eukaryotes, it has lost the stacked organisation in several lineages, raising the question of what range of morphologies is possible for the Golgi. In order to understand this diversity, it is necessary to characterise the Golgi in many different lineages. Here, we identify the Golgi complex in Naegleria, one of the first descriptions of an unstacked Golgi organelle in a non-parasitic eukaryote, other than fungi. We provide a comprehensive list of Golgi-associated membrane trafficking genes encoded in two species of Naegleria and show that nearly all are expressed in mouse-passaged N. fowleri cells. We then study distribution of the Golgi marker (Ng)CopB by fluorescence in Naegleria gruberi, identifying membranous structures that are disrupted by Brefeldin A treatment, consistent with Golgi localisation. Confocal and immunoelectron microscopy reveals that NgCOPB localises to tubular membranous structures. Our data identify the Golgi organelle for the first time in this major eukaryotic lineage, and provide the rare example of a tubular morphology, representing an important sampling point for the comparative understanding of Golgi organellar diversity.This article has an associated First Person interview with the first author of the paper.

Incidence, Pattern and Clinical Correlation of Bone Marrow Involvement In Adult T-Cell Leukaemia/Lymphoma (ATLL)

AbstractAbstract 5085 Introduction:Adult T-cell leukaemia/lymphoma (ATLL) is an aggressive T-cell lymphoma associated with the retrovirus human T-cell lymphotropic virus type 1 (HTLV-1). Cases are classified into one of four subtypes – acute, lymphomatous, chronic and smouldering - according to the criteria of Shimoyama. Essential data necessary for accurate classification are the absolute lymphocyte count, percentage of abnormal lymphocytes, corrected calcium, lactate dehydrogenase measurement and pattern of tissue involvement but assessment of bone marrow (BM) is not essential for classification. Reported median survival in aggressive ATLL is 6.2 months for acute and 10.2 months for lymphomatous subtypes. Data regarding BM involvement in ATLL is scarce coming from small series or case reports and ATLL subtype is rarely reported. In one study BM involvement occurred in 35% of patients with aggressive ATLL, subtype not specified, and was an independent predictor of poor overall survival (Takatsuki et al,2007). Aim:To determine the frequency, pattern and morphological features of BM involvement in ATLL. Methods:A retrospective analysis of pathology records of patients with ATLL diagnosed between 2000 and 2009 in whom bone marrow staging by trephine examination was undertaken. In addition to morphological analysis immunohistochemistry (IHC) was performed using a panel comprising monoclonal antibodies against CD2/CD3/CD4/CD5/CD7/CD8/CD20/CD25/CD30/CD56. CD25 staining was further analysed as membranous, cytoplasmic and golgi localisation. HTLV-1 infection was confirmed serologically in all cases. Results:Of the 19 cases 10 had lymphomatous, 8 acute and 1 chronic ATLL. The median age was 49.4 years (range 31.2–74.5) and 11 cases were female. BM involvement was detected in 12 cases (63%). This was evident morphologically in 10 patients, including all 9 with leukemic ATLL and 1 with lymphomatous disease. Disease bulk ranged from 5–40% overall but was <20% in 4/8 with acute ATLL. In 2 additional patients, both ATLL lymphoma, BM involvement was confirmed on IHC only but was minimal (<1%). The absolute lymphocyte count was significantly higher in cases with BM involvement (10.2×109/L v 1.2×109/L, p=0.004) but there was no correlation between degree of BM involvement and lymphocyte count. There was no significant difference in haemoglobin, platelet count, corrected calcium or lactate dehydrogenase levels. The pattern of infiltration was a mixture of interstitial (80%) and nodular (90%) with juxta-trabecular (30%) and peri-sinusoidal (10%) infiltrates also seen. Cell size varied from small to large with large lymphocytes more frequent in acute ATLL (75% of cases). Nuclear irregularity was present in 3/10, all of whom had a range of small, medium and large lymphocytes present. BM eosinophilia was present in 4/19 cases, all with acute ATLL. Of the 5 patients with hypercalcaemia at presentation, all with acute ATLL, bone resorption was observed in 2/5. On IHC the morphologically abnormal cells were CD3+ (11/12), CD2+ (9/10) and CD4+ (9/10). One case was CD4/CD8 dual positive. CD25 was positive in 100% and staining was a mixture of membranous (100%), cytoplasmic (89%) and golgi (22%). CD7 and CD56 were negative in all cases. CD5 expression was lost in 2/8 cases. CD30 was positive on large cells only and this was noted in 2/7 cases. Overall median survival was 8.5 months (range 0.4–72.5) and outcome did not differ according to the presence of bone marrow disease (BM+ 8.0 v BM- 10.4 months, p=0.33). Conclusion:BM involvement was present in all cases with leukaemic ATLL but only in a minority with lymphomatous ATLL, in 2 cases detectable by IHC only; the latter suggests the usefulness of this technique in residual disease assessment post therapy. Appearances were heterogenous with nodular and interstitial infiltrates frequently present in the same case. Eosinophilia and bone resorption were present in cases of acute ATLL only. The significance of BM involvement in ATLL prognosis remains uncertain. Disclosures:No relevant conflicts of interest to declare.

Localisation and Mislocalisation of the Interferon-Inducible Immunity-Related GTPase, Irgm1 (LRG-47) in Mouse Cells

Irgm1 (LRG-47) is an interferon-inducible Golgi membrane associated GTPase of the mouse whose disruption causes susceptibility to many different intracellular pathogens. Irgm1 has been variously interpreted as a regulator of homologous effector GTPases of the IRG family, a regulator of phagosome maturation and as an initiator of autophagy in interferon-induced cells. We find that endogenous Irgm1 localises to late endosomal and lysosomal compartments in addition to the Golgi membranes. The targeting motif known to be required for Golgi localisation is surprisingly also required for endolysosomal localisation. However, unlike Golgi localisation, localisation to the endolysosomal system also requires the functional integrity of the nucleotide binding site, and thus probably reflects transient activation. Golgi localisation is lost when Irgm1 is tagged at either N- or C-termini with EGFP, while localisation to the endolysosomal system is relatively favoured. N-terminally tagged Irgm1 localises predominantly to early endosomes, while C-terminally tagged Irgm1 localises to late endosomes and lysosomes. Both these anomalous distributions are reversed by inactivation of the nucleotide binding site, and the tagged proteins both revert to Golgi membrane localisation. Irgm1 is the first IRG protein to be found associated with the endolysosomal membrane system in addition to either Golgi (Irgm1 and Irgm2) or ER (Irgm3) membranes, and we interpret the result to be in favour of a regulatory function of IRGM proteins at cellular membrane systems. In future analyses it should be borne in mind that tagging of Irgm1 leads to loss of Golgi localisation and enhanced localisation on endolysosomal membranes, probably as a result of constitutive activation.

Golgi localisation of GMAP210 requires two distinct cis-membrane binding mechanisms

BackgroundThe Golgi apparatus in mammals appears as a ribbon made up of interconnected stacks of flattened cisternae that is positioned close to the centrosome in a microtubule-dependent manner. How this organisation is achieved and retained is not well understood. GMAP210 is a long coiled-coil cis-Golgi associated protein that plays a role in maintaining Golgi ribbon integrity and position and contributes to the formation of the primary cilium. An amphipathic alpha-helix able to bind liposomes in vitro has been recently identified at the first 38 amino acids of the protein (amphipathic lipid-packing sensor motif), and an ARF1-binding domain (Grip-related Arf-binding domain) was found at the C-terminus. To which type of membranes these two GMAP210 regions bind in vivo and how this contributes to GMAP210 localisation and function remains to be investigated.ResultsBy using truncated as well as chimeric mutants and videomicroscopy we found that both the N-terminus and the C-terminus of GMAP210 are targeted to the cis-Golgi in vivo. The ALPS motif was identified as the N-terminal binding motif and appeared concentrated in the periphery of Golgi elements and between Golgi stacks. On the contrary, the C-terminal domain appeared uniformly distributed in the cis-cisternae of the Golgi apparatus. Strikingly, the two ends of the protein also behave differently in response to the drug Brefeldin A. The N-terminal domain redistributed to the endoplasmic reticulum (ER) exit sites, as does the full-length protein, whereas the C-terminal domain rapidly dissociated from the Golgi apparatus to the cytosol. Mutants comprising the full-length protein but lacking one of the terminal motifs also associated with the cis-Golgi with distribution patterns similar to those of the corresponding terminal end whereas a mutant consisting in fused N- and C-terminal ends exhibits identical localisation as the endogenous protein.ConclusionWe conclude that the Golgi localisation of GMAP210 is the result of the combined action of the two N- and C-terminal domains that recognise different sub-regions of the cis-GA. Based on present and previous data, we propose a model in which GMAP210 would participate in homotypic fusion of cis-cisternae by anchoring the surface of cisternae via its C-terminus and projecting its distal N-terminus to bind the rims or to stabilise tubular structures connecting neighbouring cis-cisternae.

COP-binding sites in p24δ 2 are necessary for proper secretory cargo biosynthesis

The p24 family is thought to be somehow involved in endoplasmic reticulum-to-Golgi protein transport, and its members are major constituents of transport vesicles and bind to the vesicle coat protein complexes COPI and COPII. A subset of the p24 proteins (p24α 3, -β 1, -γ 3 and -δ 2) is upregulated when Xenopus laevis intermediate pituitary melanotrope cells are physiologically activated to produce vast amounts of their major secretory cargo, the prohormone proopiomelanocortin (POMC). To investigate the role of the COP-binding motifs of p24 proteins in POMC biosynthesis, we here generated and analysed Xenopus with stable, melanotrope cell-specific transgene expression of p24δ 2-GFP mutated in its COPI- or COPII-binding motif. In contrast to what has been found previously for wild-type (wt) p24δ 2-GFP, the p24δ 2 mutations prevented the Golgi localisation of the transgene products and caused a reduced rate of POMC cleavage, but did not lead to a reduction of the endogenous p24 proteins nor to aberrations in POMC glycosylation and sulphation. We conclude that p24δ 2 requires the presence of the COPI- and COPII-binding sites to allow proper POMC processing. Thus, the p24 proteins fulfil their role in secretory protein biosynthesis via COPI- or COPII-coated transport vesicles.

The cytoplasmic and transmembrane domains of secretor type α1,2fucosyltransferase confer atypical cellular localisation

Carbohydrate structures influence many aspects of cell biology. Manipulating the glycosyltransferase enzymes, that sequentially add carbohydrate moieties to proteins and lipids as they pass through the Golgi and secretory pathway, can alter these carbohydrate epitopes. We previously demonstrated that the eight amino acid cytoplasmic tail of alpha1,2fucosyltransferase (FT) contained a sequence for Golgi localisation. In this study, we examined the localisation of the closely related secretor type alpha1,2fucosyltransferase (Sec) which has a smaller, yet apparently unrelated, five amino acid cytoplasmic tail. In contrast to the Golgi localisation of FT, Sec displayed atypical cytoplasmic vesicular-like staining. However, replacing just the five amino acid tail of Sec with FT was sufficient to relocalise the enzyme to a perinuclear region with Golgi-like staining. The biological significance of this relocalisation was this chimaeric enzyme was more effective than FT at competing for N-Acetyl-lactosamine and thus was superior in reducing expression of the Galalpha(1,3)Gal xenoepitope.

Phosphorylation at Ser729 specifies a Golgi localisation for protein kinase C epsilon (PKCε) in 3T3 fibroblasts

We demonstrate that GFP-PKCε concentrates at a perinuclear site in living fibroblasts and that cell passage induces rapid translocation of PKCε to the periphery where it appears to colocalise with F-actin. When newly passaged cells have adhered and are proliferating again, GFP-PKCε returns to its perinuclear site. GFP-PKCε co-localises with wheat germ agglutinin suggesting that it is associated with the Golgi at the perinuclear site. In support, PKCε is detected in a Golgi-enriched fraction in pre-passage cells but is lost from the fraction after passage. PKCε at the perinuclear Golgi site is phosphorylated at Ser729 but cell passage induces the loss of the phosphate at this site as reported previously [England et al. (2001) J. Biol. Chem. 276, 10437-10442]. PKCε S729A, S729E and S729T mutants, which are not recognised by a specific antiphosphoPKCε (Ser729) antibody, do not concentrate at a perinuclear/Golgi site in proliferating fibroblasts. This suggests that both phosphorylation and serine rather than threonine are needed at position 729 to locate PKCε at its perinuclear/Golgi site. Phorbol ester induced translocation of PKCε to the nucleus also requires dephosphorylation at Ser729; after translocation nuclear PKCε lacks a phosphate at Ser729. Sulphation and secretion of glycosaminoglycan (GAG) chains from fibroblasts increases on passage and returns to basal as cells proliferate showing that cell passage influences secretory events at the Golgi. The results indicate that Ser729 phosphorylation plays a role in determining PKCε localisation in fibroblasts.

The cytoplasmic domain of tomato spotted wilt virus Gn glycoprotein is required for Golgi localisation and interaction with Gc

Envelopment of tomato spotted wilt virus nucleocapsids occurs at the Golgi stacks of infected cells. This is also the place where the two membrane glycoproteins Gn and Gc accumulate upon coexpression. The required Golgi retention signal has previously been demonstrated to reside within Gn. Using a series of truncated Gn proteins, the Golgi retention signal was mapped to a stretch of 10 amino acids on this protein's cytoplasmic tail, 20 residues downstream the transmembrane domain. Studies on the intracellular distribution of chimeric Gc proteins in which the cytoplasmic tail and/or transmembrane domain were exchanged by those from Gn, demonstrated the additional requirement of the Gn transmembrane domain for Golgi targeting. Truncated Gn constructs lacking the C-terminal 20 amino acids but still localising to the Golgi were no longer able to redirect Gc, suggesting the requirement of this domain for interaction with Gc.

Human TPST1 Transmembrane Domain Triggers Enzyme Dimerisation and Localisation to the Golgi Compartment

TPST1 is a human tyrosylprotein sulfotransferase that uses 3′phosphoadenosine-5′phosphosulfate (PAPS) to transfer the sulfate moiety to proteins predominantly designated for secretion. To achieve a general understanding of the cellular role of human tyrosine-directed sulfotransferases, we investigated targeting, structure and posttranslational modification of TPST1. Golgi localisation of the enzyme in COS-7 and HeLa cells was visualised by fluorescence imaging techniques. PNGase treatment and mutational studies determined that TPST1 bears N-linked glycosyl residues exclusively at position Asn60 and Asn262. By alanine mutation of these asparagine residues, we could determine that the N-linked oligosaccharides do not have an influence on Golgi retention of TPST1. In concert with N and C-terminal flanking residues, the transmembrane domain of TPST1 was determined to act in targeting and retention of the enzyme to the trans-Golgi compartment. This domain exhibits a pronounced secondary structure in a lipid environment. Further in vivo FRET studies using the transmembrane domain suggest that the human tyrosylprotein sulfotransferase may be functional as homodimer/oligomer in the trans-Golgi compartment.

The regulatory domain of protein kinase Ctheta localises to the Golgi complex and induces apoptosis in neuroblastoma and Jurkat cells.

This study investigates apoptotic effects of protein kinase C (PKC) delta and theta in neuroblastoma cells. 12-O-tetradecanoylphorbol-13-acetate induces apoptosis in SK-N-BE(2) neuroblastoma cells overexpressing PKCdelta or PKCtheta, but not PKC epsilon. The PKC inhibitor GF109203X does not suppress this apoptotic effect, suggesting that it is independent of the catalytic activity of PKC. The isolated catalytic domains of PKCdelta and PKCtheta or the regulatory domain (RD) of PKCtheta also induce apoptosis in neuroblastoma cells. The apoptotic responses are suppressed by caspase inhibition and by Bcl-2 overexpression. The PKCtheta RD induced apoptosis also in Jurkat cells. Colocalisation analysis revealed that the PKCtheta RD primarily localises to the Golgi complex. The C1b domain is required for this localisation and removal of the C1b domain results in a PKCtheta construct that does not induce apoptosis. This suggests that the PKCtheta RD has apoptotic activity and that Golgi localisation may be important for this effect.

The notch signalling regulator fringe acts in the Golgi apparatus and requires the glycosyltransferase signature motif DXD.

Signalling via the Notch receptor is a key regulator of many developmental processes. The differential responsiveness of Notch-expressing cells to the ligands Delta and Serrate is controlled by Fringe, itself essential for normal patterning in Drosophila and vertebrates. The mechanism of Fringe action, however, is not known. The protein has an amino-terminal hydrophobic stretch resembling a cleaved signal peptide, which has led to the widespread assumption that it is a secreted signalling molecule. It also has distant homology to bacterial glycosyltransferases, although it is not clear if this reflects a shared enzymatic activity, or merely a related structure. We report that a functional epitope-tagged form of Drosophila Fringe was localised in the Golgi apparatus. When the putative signal peptide was replaced by a confirmed one, Fringe no longer accumulated in the Golgi, but was instead efficiently secreted. This change in localisation dramatically reduced its biological activity, implying that the wild-type protein normally acts inside the cell. We show that Fringe specifically binds the nucleoside diphosphate UDP, a feature of many glycosyltransferases. Furthermore, specific mutation of a DxD motif (in the single-letter amino acid code where x is any amino acid), a hallmark of most glycosyltransferases that use nucleoside diphosphate sugars, did not affect the Golgi localisation of the protein but completely eliminated in vivo activity. These results indicate that Fringe does not exert its effects outside of the cell, but rather acts in the Golgi apparatus, apparently as a glycosyltransferase. They suggest that alteration in receptor glycosylation can regulate the relative efficiency of different ligands.

Overexpression of the alpha2,6 (N) sialyltransferase enzyme in human and rat neural cell lines is associated with increased expression of the polysialic acid epitope.

The function of the neural cell adhesion molecule, NCAM, is modulated by the expression of the N-linked polysialic acid (PSA) oligosaccharide chain, with PSA serving to decrease the adhesive potential of the protein backbone. In this study, we have generated clonal cells of the rat B104 and human SH-SY5Y neuroblastoma cell lines that over-express the alpha2,6(N) sialyltransferase (ST6N) enzyme in order to investigate the role of this enzyme in PSA biosynthesis. The clonal cells exhibited ST enzyme activities of up to 20-times control levels, which remained stable throughout the duration of the study. The increase in enzyme activity paralleled an increase in enzyme protein levels, as determined by Western blot analysis, and immunocytochemical analysis confirmed the Golgi localisation of the enzyme. The induction of PSA-NCAM expression in the cells expressing high levels of ST6N was confirmed both by using anti-PSA antisera and by specific digestion with endo-N-acetylneuraminidase E, whose actions are specific for alpha2, 8-linked PSA chains. These results demonstrate that the cellular ST6N activity serves to positively influence the expression of PSA in neuronal cells.

A novel Golgi-localisation domain shared by a class of coiled-coil peripheral membrane proteins

The mechanism by which peripheral membrane proteins are targeted to the cytoplasmic face of the Golgi apparatus is poorly understood. Previously, we have identified a carboxy-terminal domain of the trans-Golgi-network (TGN) protein p230 that is responsible for Golgi localisation [1]. Here, we report the identification of a similar Golgi-localisation domain (GLD, also termed the ‘GRIP’ domain – see the paper by Munro and Nichols elsewhere in this issue) in a family of putative peripheral membrane proteins from lower and higher eucaryotes. The majority of family members have a domain structure similar to that of p230, with extensive coiled-coil regions (>80%) and the potential GLD located in a non-coiled-coil domain at the carboxyl terminus. Previously reported proteins in this family include human golgin-97 and Saccharomyces cerevisiae Imh1p. By constructing chimeric cDNAs encoding carboxy-terminal regions of these family members fused to green fluorescent protein (GFP), we have directly demonstrated that the GLD of p230, golgin-97, the newly identified human protein GCC1p and yeast Imh1p functions as a Golgi-targeting domain in transfected mammalian cells. Site-directed mutagenesis of the GLDs identified two conserved aromatic residues that are critical for the function of this targeting domain. Endogenous p230 was displaced from the Golgi membranes in transfected cells expressing high levels of GFP fused to the GLD of either p230 or golgin-97, indicating that different GLDs interact with similar membrane determinants. Thus, we have identified a family of coiled-coil proteins that share a domain shown to be sufficient for the localisation of peripheral membrane proteins to the Golgi apparatus.

Growth hormone receptor expression in the nucleus and cytoplasm of normal and neoplastic cells.

Growth hormone (GH) exerts its regulatory functions in controlling metabolism, balanced growth and differentiated cell expression by acting on specific receptors which trigger a phosphorylation cascade, resulting in the modulation of numerous signalling pathways dictating gene expression. A panel of five monoclonal antibodies was used in mapping the presence and somatic distribution of the GH receptor by immunohistochemistry in normal and neoplastic tissues and cultured cells of human, rat and rabbit origin. A wide distribution of the receptor was observed in many cell types. Not all cells expressing cytoplasmic GH receptors displayed nuclear immunoreactivity. In general, the relative proportion of positive cells and intensity of staining was higher in neoplastic cells than in normal tissue cells. Immunoreactivity showed subcellular localisation of the GH receptor in cell membranes and was predominantly cytoplasmic, but strong nuclear immunoreaction was also apparent in many instances. Intense immunoreactivity was also observed in the cellular Golgi area of established cell lines and cultured tissue-derived cells in exponential growth phase, indicating cells are capable of GH receptor synthesis. The presence of intracellular GH receptor, previously documented in normal tissues of mostly animal origin, is the result of endoplasmic reticulum and Golgi localisation. Heterogeneity of immunoreactivity was found in normal and neoplastic tissue with a variable range of positive cells. The nuclear localisation of immunoreactivity is the result of nuclear GH receptor/binding protein, identically to the cytosolic and plasma GH-binding protein, using a panel of five monoclonal antibodies against the GH receptor extracellular region. The expression of GH receptors, not only on small proliferating tumour cells such as lymphocytes, but also on well differentiated cells including keratinocytes, suggests that GH is necessary not only for differentiation of progenitor cells, but also for their subsequent clonal expansion, differentiation and maintenance.