Increased LAMP-2 polylactosamine glycosylation is associated with its slower Golgi transit during establishment of a polarized MDCK epithelial monolayer.

An endogenous Madin-Darby canine kidney (MDCK) lysosomal membrane glycoprotein that exhibits a basolateral targeting pathway to the lysosome is shown here to exhibit significant N-terminal amino acid sequence identity to lysosomal associated membrane proteins (LAMP-2) of other species. During establishment of the MDCK monolayer after only 1 d of culture, this canine LAMP-2 has a larger molecular size (110 kDa) than following formation of a confluent monolayer after 3 d of culture (100 kDa) due to the increased presence of N-linked polylactosamine oligosaccharide chains. Neither polylactosamine glycosylation of LAMP-2 in MDCK cells nor truncation of N-linked oligosaccharide chains of LAMP-2 in a ricin-resistant MDCK-RCAR cell line influenced the basolateral polarity of its targeting. However, the rate of basolateral delivery of LAMP-2 in MDCK cells plated for 3 d was significantly faster (t1/2 = 28 min) than in 1-d cells (t1/2 = 40 min); in MDCK-RCAR cells the rate of basolateral delivery at both 1 and 3 d of plating was similar (t1/2 = 40 min). The rate differential in MDCK cells occurred after arrival of LAMP-2 to the Golgi apparatus because the rate of acquisition of endoglycosidase H resistance was the same (t1/2 = 25 min) at both days of plating. The rate of transit of LAMP-2 through the Golgi apparatus to the basolateral domain was therefore far more rapid (approximately 4-fold) in 3 d compared with 1-d MDCK cultures. The increased polylactosamine glycosylation of MDCK LAMP-2 at early times of plating during the establishment of a confluent epithelial monolayer may thus be related to its longer residence time in the Golgi apparatus.     

The cytosolic phospholipase A2 catalytic domain modulates association and residence time at Golgi membranes

Cytosolic phospholipase A2 (cPLA2) catalyzes release of arachidonic acid from membranes following translocation to Golgi and endoplasmic reticulum. In response to an intracellular calcium concentration ([Ca2+]i) increase, the C2 domain binds Ca2+ and brings the catalytic domain into proximity with its phospholipid substrate. Because membrane residence is important in the regulation of cPLA2 activity, we explored the contributions of the C2 and catalytic domains in mediating membrane residence using an imaging approach in live cells with fluorescent protein chimeras of cPLA2. The isolated cPLA2 C2 domain associated with Golgi membranes rapidly in proportion to the [Ca2+]i, allowing for its use as a [Ca2+]i indicator. cPLA2 association with Golgi was slower than the isolated C2 domain in response to a [Ca2+]i increase. After [Ca2+]i decrease, cPLA2 remained associated with membrane in a Ca(2+)-independent fashion whereas C2 domain rapidly dissociated. Ca(2+)-independent membrane association was greatly reduced by mutation of Trp464, located at the membrane-exposed face of the catalytic domain, to Gly or Ala. Mutation of Trp464 to Phe supported Ca(2+)-independent association similar to wild type. These results demonstrate a role for the cPLA2 catalytic domain in regulating membrane association and membrane residence time.     

Cytotoxicity of gelonin and its conjugates with antibodies is determined by the extent of their endocytosis

Conjugates of the single-chain ribosome-inactivating protein gelonin with ligands that bind to cell surface molecules vary greatly in their cytotoxicity. Conjugates that are not endocytosed after binding to cells exhibit low cytotoxicity similar to that of free gelonin, while conjugates that are endocytosed demonstrate enhanced cytotoxicity relative to free gelonin. However, the number of internalized gelonin molecules needed to intoxicate cells to the same degree has been found to be similar for all conjugates and for free gelonin. The intracellular concentration of gelonin has to be between 2,000-10,000 molecules/cells to achieve a surviving fraction of 0.37. Our studies revealed the presence of three distinct categories of cell surface molecules, those that are efficient in mediating endocytosis of immunotoxins, those that are only moderately efficient, and those that seem not to cause internalization of bound immunotoxins.     

The Golgi localization of GOLPH2 (GP73/GOLM1) is determined by the transmembrane and cytoplamic sequences

Golgi phosphoprotein 2 (GOLPH2) is a resident Golgi type-II membrane protein upregulated in liver disease. Given that GOLPH2 traffics through endosomes and can be secreted into the circulation, it is a promising serum marker for liver diseases. The structure of GOLPH2 and the functions of its different protein domains are not known. In the current study, we investigated the structural determinants for Golgi localization using a panel of GOLPH2 truncation mutants. The Golgi localization of GOLPH2 was not affected by the deletion of the C-terminal part of the protein. A truncated mutant containing the N-terminal portion (the cytoplasmic tail and transmembrane domain (TMD)) localized to the Golgi. Sequential deletion analysis of the N-terminal indicated that the TMD with a positively charged residue in the cytoplasmic N-terminal tail were sufficient to support Golgi localization. We also showed that both endogenous and secreted GOLPH2 exist as a disulfide-bonded dimer, and the coiled-coil domain was sufficient for dimerization. This structural knowledge is important for the understanding the pathogenic role of GOLPH2 in liver diseases, and the development of GOLPH2-based hepatocellular cancer diagnostic methods.     

COG complex-mediated recycling of Golgi glycosyltransferases is essential for normal protein glycosylation

Defects in conserved oligomeric Golgi (COG) complex result in multiple deficiencies in protein glycosylation. On the other hand, acute knock-down (KD) of Cog3p (COG3 KD) causes accumulation of intra-Golgi COG complex-dependent (CCD) vesicles. Here, we analyzed cellular phenotypes at different stages of COG3 KD to uncover the molecular link between COG function and glycosylation disorders. For the first time, we demonstrated that medial-Golgi enzymes are transiently relocated into CCD vesicles in COG3 KD cells. As a result, Golgi modifications of both plasma membrane (CD44) and lysosomal (Lamp2) glycoproteins are distorted. Localization of these proteins is not altered, indicating that the COG complex is not required for anterograde trafficking and accurate sorting. COG7 KD and double COG3/COG7 KD caused similar defects with respect to both Golgi traffic and glycosylation, suggesting that the entire COG complex orchestrates recycling of medial-Golgi-resident proteins. COG complex-dependent docking of isolated CCD vesicles was reconstituted in vitro, supporting their role as functional trafficking intermediates. Altogether, the data suggest that constantly cycling medial-Golgi enzymes are transported from distal compartments in CCD vesicles. Dysfunction of COG complex leads to separation of glycosyltransferases from anterograde cargo molecules passing along secretory pathway, thus affecting normal protein glycosylation.     

The irreversibility of inner mitochondrial membrane permeabilization by Ca2+ plus prooxidants is determined by the extent of membrane protein thiol cross-linking

We have previously shown that mitochondrial membrane potential () drop promoted by prooxidants and Ca²⁺ can be reversed but not sustained by ethylene glycol-bis(-aminoethylether)-N,N,N,N-tetraacetic acid (EGTA) unless dithiothreitol (DTT), a disulfide reductant, is also added [Valle, V. G. R., Fagian, M. M., Parentoni, L. S., Meinicke, A. R., and Vercesi, A. E. (1993).Arch. Biochem. Biophys. 307, 1–7]. In this study we show that catalase or ADP are also able to potentiate this EGTA effect. When EGTA is added long after (12 min) the completion of swelling or elimination, no membrane resealing occurs unless the EGTA addition was preceded by the inclusion of DTT, ADP, or catalase soon after was collapsed. Total recovery by EGTA is obtained only in the presence of ADP. The sensitivity of the ADP effect to carboxyatractyloside strongly supports the involvement of the ADP/ATP carrier in this mechanism. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized membrane proteins shows that protein aggregation due to thiol cross-linkage formed during drop continues even after is already eliminated. Titration with 5,5-dithio-bis(2-nitrobenzoic acid) supports the data indicating that the formation of protein aggregates is paralleled by a decrease in the content of membrane protein thiols. Since the presence of ADP and EGTA prevents the progress of protein aggregation, we conclude that this process is responsible for both increased permeability to larger molecules and the irreversibility of drop. The protective effect of catalase suggests that the continuous production of protein thiol cross-linking is mediated by mitochondrial generated reactive oxygen species.     

Elevated Golgi pH impairs terminal N‐glycosylation by inducing mislocalization of Golgi glycosyltransferases

Acidic pH of the Golgi lumen is known to be crucial for correct glycosylation, transport and sorting of proteins and lipids during their transit through the organelle. To better understand why Golgi acidity is important for these processes, we have examined here the most pH sensitive events in N‐glycosylation by sequentially raising Golgi luminal pH with chloroquine (CQ), a weak base. We show that only a 0.2 pH unit increase (20 µM CQ) is sufficient to markedly impair terminal α(2,3)‐sialylation of an N‐glycosylated reporter protein (CEA), and to induce selective mislocalization of the corresponding α(2,3)‐sialyltransferase (ST3) into the endosomal compartments. Much higher pH increase was required to impair α(2,6)‐sialylation, or the proximal glycosylation steps such as β(1,4)‐galactosylation or acquisition of Endo H resistance, and the steady‐state localization of the key enzymes responsible for these modifications (ST6, GalT I, MANII). The overall Golgi morphology also remained unaltered, except when Golgi pH was raised close to neutral. By using transmembrane domain chimeras between the ST6 and ST3, we also show that the luminal domain of the ST6 is mainly responsible for its less pH sensitive localization in the Golgi. Collectively, these results emphasize that moderate Golgi pH alterations such as those detected in cancer cells can impair N‐glycosylation by inducing selective mislocalization of only certain Golgi glycosyltransferases. J. Cell. Physiol. 220: 144–154, 2009. © 2009 Wiley‐Liss, Inc.     

Clathrin-mediated endocytosis of MUC1 is modulated by its glycosylation state

MUC1 is a mucin-like type 1 transmembrane protein associated with the apical surface of epithelial cells. In human tumors of epithelial origin MUC1 is overexpressed in an underglycosylated form with truncated O-glycans and accumulates in intracellular compartments. To understand the basis for this altered subcellular localization, we compared the synthesis and trafficking of various glycosylated forms of MUC1 in normal (Chinese hamster ovary) cells and glycosylation-defective (ldlD) cells that lack the epimerase to make UDP-Gal/GalNAc from UDP-Glc/GlcNAc. Although the MUC1 synthesized in ldlD cells was rapidly degraded, addition of GalNAc alone to the culture media resulted in stabilization and near normal surface expression of MUC1 with truncated but sialylated O-glycans. Interestingly, the initial rate of endocytosis of this underglycosylated MUC1 was stimulated by twofold compared with fully glycosylated MUC1. However, the half-lives of the two forms were not different, indicating that trafficking to lysosomes was not affected. Both the normal and stimulated internalization of MUC1 could be blocked by hypertonic media, a hallmark of clathrin-mediated endocytosis. MUC1 endocytosis was also blocked by expression of a dominant-negative mutant of dynamin-1 (K44A), and MUC1 was observed in both clathrin-coated pits and vesicles by immunoelectron microscopy of ultrathin cryosections. Our data suggest that the subcellular redistribution of MUC1 in tumor cells could be a direct result of altered endocytic trafficking induced by its aberrant glycosylation; potential models are discussed. These results also implicate a new role for O-glycans on mucin-like membrane proteins entering the endocytic pathway through clathrin-coated pits.     

The Golgi mitotic checkpoint is controlled by BARS-dependent fission of the Golgi ribbon into separate stacks in G2

The Golgi ribbon is a complex structure of many stacks interconnected by tubules that undergo fragmentation during mitosis through a multistage process that allows correct Golgi inheritance. The fissioning protein CtBP1-S/BARS (BARS) is essential for this, and is itself required for mitotic entry: a block in Golgi fragmentation results in cell-cycle arrest in G2, defining the 'Golgi mitotic checkpoint'. Here, we clarify the precise stage of Golgi fragmentation required for mitotic entry and the role of BARS in this process. Thus, during G2, the Golgi ribbon is converted into isolated stacks by fission of interstack connecting tubules. This requires BARS and is sufficient for G2/M transition. Cells without a Golgi ribbon are independent of BARS for Golgi fragmentation and mitotic entrance. Remarkably, fibroblasts from BARS-knockout embryos have their Golgi complex divided into isolated stacks at all cell-cycle stages, bypassing the need for BARS for Golgi fragmentation. This identifies the precise stage of Golgi fragmentation and the role of BARS in the Golgi mitotic checkpoint, setting the stage for molecular analysis of this process.     

The catalytic effect of dihydrofolate reductase and its mutants is determined by reorganization energies

The effect of distant mutations on the catalytic reaction of dihydrofolate reductase (DHFR) is reexamined by empirical valence bond simulations. The simulations reproduce for the first time the changes in the observed rate constants (without the use of adjustable parameters for this purpose) and show that the changes in activation barriers are strongly correlated with the corresponding changes in the reorganization energy. The preorganization of the polar groups of enzymes is the key catalytic factor, and anticatalytic mutations destroy this preorganization. Some anticatalytic mutations in DHFR also increase the distance between the donor and acceptor, but this effect is not directly related to catalysis since the native enzyme and the uncatalyzed reaction in water have similar average donor-acceptor distances. Insight into the effect of a mutation is provided by constructing the relevant free energy surfaces in terms of the generalized solute-solvent coordinates. It is shown how the mutations change the reaction coordinate and the activation barrier, and it is clarified that the corresponding changes do not reflect dynamical effects. It is also pointed out that all reactions in a condensed phase involve correlated motions (both in enzymes and in solution) and that the change of such motions upon mutations is a result of the change in the shape of the multidimensional reaction path on the solute-solvent surface, rather than the reason for the change in rate constant. Thus, as far as catalysis is concerned, the change in the activation barrier is due to the change in the electrostatic preorganization energy.     

MEK and Cdc2 kinase are sequentially required for Golgi disassembly in MDCK cells by the mitotic Xenopus extracts

At the onset of mitosis, the Golgi apparatus, which consists of several cisternae, disperses throughout the cell to be partitioned into daughter cells. The molecular mechanisms of this process are now beginning to be understood. To investigate the biochemical requirements and kinetics of mitotic Golgi membrane dynamics in polarized cells, we have reconstituted the disassembly of the Golgi apparatus by introducing Xenopus egg extracts into permeabilized Mardin-Darby canine kidney (MDCK) cells. We used green fluorescence protein (GFP)-tagged galactosyltransferase-expressing MDCK cells to analyze the morphological changes of the Golgi membrane in the semi-intact system. Analyses by fluorescence and electron microscopies showed that the Golgi disassembly can be dissected into two elementary processes morphologically. In the first process, the perinuclear Golgi stacks break into punctate structures, intermediates, which are comprised of mini-stacks of cisternae associating with apical microtubule networks. In the second process, the structures fragment more thoroughly or substantially relocate to the ER. Our analyses further showed that cdc2 kinase and mitogen-activated protein kinase kinase (MAPKK = MEK) are differently involved in these two processes: the first process is mainly regulated by MEK and the second mainly by cdc2.     

Guanosine diphosphatase is required for protein and sphingolipid glycosylation in the Golgi lumen of Saccharomyces cerevisiae

Current models for nucleotide sugar use in the Golgi apparatus predict a critical role for the lumenal nucleoside diphosphatase. After transfer of sugars to endogenous macromolecular acceptors, the enzyme converts nucleoside diphosphates to nucleoside monophosphates which in turn exit the Golgi lumen in a coupled antiporter reaction, allowing entry of additional nucleotide sugar from the cytosol. To test this model, we cloned the gene for the S. cerevisiae guanosine diphosphatase and constructed a null mutation. This mutation should reduce the concentrations of GDP-mannose and GMP and increase the concentration of GDP in the Golgi lumen. The alterations should in turn decrease mannosylation of proteins and lipids in this compartment. In fact, we found a partial block in O- and N-glycosylation of proteins such as chitinase and carboxypeptidase Y and underglycosylation of invertase. In addition, mannosylinositolphosphorylceramide levels were drastically reduced.     

Rapid turnover of c-FLIPshort is determined by its unique C-terminal tail

The caspase-8 inhibitor c-FLIP exists as two splice variants, c-FLIP(L) and c-FLIP(S), with distinct roles in death receptor signaling. The mechanisms determining their turnover have not been established. We found that in differentiating K562 erythroleukemia cells both c-FLIP isoforms were inducibly degraded by the proteasome, but c-FLIP(S) was more prone to ubiquitylation and had a considerably shorter half-life. Analysis of the c-FLIP(S)-specific ubiquitylation revealed two lysines, 192 and 195, C-terminal to the death effector domains, as principal ubiquitin acceptors in c-FLIP(S) but not in c-FLIP(L). Furthermore the c-FLIP(S)-specific tail of 19 amino acids, adjacent to the two target lysines, was demonstrated to be the key element determining the isoform-specific instability of c-FLIP(S). Molecular modeling in combination with site-directed mutagenesis demonstrated that the C-terminal tail is required for correct positioning and subsequent ubiquitylation of the target lysines. Because the antiapoptotic operation of c-FLIP(S) was not affected by the tail deletion, the antiapoptotic activity and ubiquitin-mediated degradation of c-FLIP(S) are functionally and structurally independent processes. The presence of a small destabilizing sequence in c-FLIP(S) constitutes an important determinant of c-FLIP(S)/c-FLIP(L) ratios by allowing differential degradation of c-FLIP isoforms. The conformation-based predisposition of c-FLIP(S) to ubiquitin-mediated degradation introduces a novel concept to the regulation of the death-inducing signaling complex.     

The stability of yeast invertase is not significantly influenced by glycosylation

Yeast invertase exists in two different forms. The cytoplasmic enzyme is nonglycosylated, whereas the external invertase contains about 50% carbohydrate of the high mannose type. The protein moieties of both enzymes are identical. The two invertases have been used previously as a model system to investigate the influence of covalently linked carbohydrate chains on the stability of large glycoproteins, and controversial results were obtained. Here, we measured thermal and denaturant-induced unfolding by various probes, such as the loss of enzymatic activity, and by the changes in absorbance and fluorescence. The ranges of stability of the two invertases were found to be essentially identical, indicating that the presence of a high amount of carbohydrate does not significantly contribute to the stability of external invertase. Earlier findings that invertase is stabilized by glycosylation could not be confirmed. The stability of this glycoprotein is apparently determined by the specific interactions of the folded polypeptide chain. Unlike the glycosylated form, the carbohydrate-free invertase is prone to aggregation in the denatured state at high temperature and in a partially unfolded form in the presence of intermediate concentrations of guanidinium chloride.     

The maximum height of grasses is determined by roots

Grasses such as bamboos can produce upright stems more than 30 m tall, yet the processes that constrain plant height in this important group have never been investigated. Air embolisms form commonly in the water transport system of grasses and we hypothesised that root pressure-dependent refilling these embolisms should limit the maximum height of grass species to the magnitude of their root pressure. Confirming this hypothesis, we show that in 59 species of bamboo grown in two common gardens, the maximum heights of culms of 67 clones are closely predicted by the maximum measured root pressure overnight. Furthermore, we demonstrate that water transport in these bamboo species is dependent on root pressure to repair hydraulic dysfunction sustained during normal diurnal gas exchange. Our results established the critical importance of root pressure in the tallest grass species and provide a new basis for understanding the limits for plant growth.     

AtCSLD2 is an integral Golgi membrane protein with its N-terminus facing the cytosol

Cellulose synthase-like proteins in the D family share high levels of sequence identity with the cellulose synthase proteins and also contain the processive beta-glycosyltransferase motifs conserved among all members of the cellulose synthase superfamily. Consequently, it has been hypothesized that members of the D family function as either cellulose synthases or glycan synthases involved in the formation of matrix polysaccharides. As a prelude to understanding the function of proteins in the D family, we sought to determine where they are located in the cell. A polyclonal antibody against a peptide located at the N-terminus of the Arabidopsis D2 cellulose synthase-like protein was generated and purified. After resolving Golgi vesicles from plasma membranes using endomembrane purification techniques including two-phase partitioning and sucrose density gradient centrifugation, we used antibodies against known proteins and marker enzyme assays to characterize the various membrane preparations. The Arabidopsis cellulose synthase-like D2 protein was found mostly in a fraction that was enriched with Golgi membranes. In addition, versions of the Arabidopsis cellulose synthase-like D2 proteins tagged with a green fluorescent protein was observed to co-localize with a DsRed-tagged Golgi marker protein, the rat alpha-2,6-sialyltransferase. Therefore, we postulate that the majority of Arabidopsis cellulose synthase-like D proteins, under our experimental conditions, are likely located at the Golgi membranes. Furthermore, protease digestion of Golgi-rich vesicles revealed almost complete loss of reaction with the antibodies, even without detergent treatment of the Golgi vesicles. Therefore, the N-terminus of the Arabidopsis cellulose synthase-like D2 protein likely faces the cytosol. Combining this observation with the transmembrane domain predictions, we postulate that the large hydrophilic domain of this protein also faces the cytosol.     

Reovirus-induced apoptosis of MDCK cells is not linked to viral yield and is blocked by Bcl-2.

In this study, we investigated the relationship between reovirus-induced apoptosis and viral growth. Madin-Darby canine kidney (MDCK) epithelial cells infected with prototype reovirus strains type 1 Lang (T1L) or type 3 Dearing (T3D) were found to undergo apoptosis, and T3D induced apoptosis of MDCK cells to a substantially greater extent than T1L. By using T1L x T3D reassortant viruses, we found that differences in the capacities of these strains to induce apoptosis are determined by the viral S1 and M2 gene segments. These genes encode viral outer-capsid proteins that play important roles in viral entry into cells. T1L grew significantly better in MDCK cells than T3D, and these differences in growth segregated with the viral L1 and M1 gene segments. The L1 and M1 genes encode viral core proteins involved in viral RNA synthesis. Bcl-2 overexpression in MDCK cells inhibited reovirus-induced apoptosis but did not substantially affect reovirus growth. These findings indicate that differences in the capacities of reovirus strains to induce apoptosis and grow in MDCK cells are determined by different viral genes and that premature cell death by apoptosis does not limit reovirus growth in MDCK cells.