Submicromolar manganese dependence of Golgi vesicular galactosyltransferase (lactose synthetase)

Kuhn, Nicholas J.; Ward, Simon; Leong, Weng S.

doi:10.1111/j.1432-1033.1991.tb15700.x

Cited by 24 publications

(13 citation statements)

References 30 publications

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“…For example, manganese needs to be delivered to the Golgi apparatus where the metal activates a variety of sugar transferases involved in protein processing (43)(44)(45)(46). In yeast, a useful marker for Golgi manganese is invertase, an enzyme that is modified by N-glycosylation via manganese-dependent mannosyltransferases (45)(46)(47)(48).…”

Section: Resultsmentioning

confidence: 99%

Manganese Superoxide Dismutase in Saccharomyces cerevisiae Acquires Its Metal Co-factor through a Pathway Involving the Nramp Metal Transporter, Smf2p

Luk¹,

Culotta²

2001

Journal of Biological Chemistry

135

166

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Eukaryotes express both copper/zinc (SOD1)-and manganese (SOD2)-requiring superoxide dismutase enzymes that guard against oxidative damage. Although SOD1 acquires its copper through a specific copper trafficking pathway, nothing is known regarding the intracellular manganese trafficking pathway for SOD2. We demonstrate here that in Saccharomyces cerevisiae cells delivery of manganese to SOD2 in the mitochondria requires the Nramp metal transporter, Smf2p. SOD2 activity is greatly diminished in smf2⌬ mutants, even though the mature SOD2 polypeptide accumulates to normal levels in mitochondria. Treating smf2⌬ cells with manganese supplements corrected the SOD2 defect, as did elevating intracellular manganese through mutations in PMR1. Hence, manganese appears to be inaccessible to mitochondrial SOD2 in smf2 mutants. Cells lacking SMF2 also exhibited defects in manganese-dependent steps in protein glycosylation and showed an overall decrease in steady-state levels of accumulated manganese. By comparison, mutations in the cell surface Nramp transporter, Smf1p, had very little impact on manganese accumulation and trafficking. Smf2p resides in intracellular vesicles and shows no evidence of plasma membrane localization, even in an end4 mutant blocked for endocytosis. We propose a model in which Smf2p-containing vesicles play a central role in manganese trafficking to the mitochondria and other cellular sites as well. Superoxide dismutases (SOD)1 are critical anti-oxidant defense enzymes that catalyze the disproportionation of superoxide anion to oxygen and hydrogen peroxide. Eukaryotic cells possess two evolutionary distinct forms of SOD: a copper-and zinc-containing SOD (SOD1) found mainly in the cytosol (1) and a manganese-containing SOD (SOD2) that localizes strictly to the mitochondrial matrix (2). Because both forms of SOD require a transition metal co-factor, enzyme activity in cells may be controlled through availability of the cognate metal ion. Indeed this is the case for the copper-requiring SOD1, where the enzyme relies on components of a defined copper trafficking pathway for acquisition of its metal ion (for reviews see Refs. 3-6). However, nothing is known regarding the mechanism by which SOD2 receives its manganese cofactor in vivo.Encoded in the nucleus, SOD2 is predicted to acquire its manganese after import into the mitochondrial matrix. The manganese-binding residues of SOD2 occur at distant regions of the polypeptide (7), and the metal binding site is not likely to remain intact during the protein-unfolding process needed for mitochondrial membrane translocation. Therefore, an intricate manganese trafficking pathway must warrant the accurate delivery of the metal from the cell surface to the mitochondria. To date, none of the factors that participate in this pathway have been identified.The bakers' yeast Saccharomyces cerevisiae represents an ideal eukaryotic system in which to unravel metal trafficking pathways. This organism has been extremely powerful for elucidating the copper acquisition pathw...

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Section: Resultsmentioning

confidence: 99%

Manganese Superoxide Dismutase in Saccharomyces cerevisiae Acquires Its Metal Co-factor through a Pathway Involving the Nramp Metal Transporter, Smf2p

Luk¹,

Culotta²

2001

Journal of Biological Chemistry

135

166

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“…Um Komplikationen, die durch das paramagnetische Verhalten von Mn 2+ zu erwarten wären, zu unterbinden, wurde Mn 2+ durch Mg 2+ im Puffer ersetzt. Die Enzymaktivität wird durch Mg 2+ zwar reduziert, die Funktion des Proteins bleibt aber erhalten 7. Von dieser Probe wurden 1D‐STD‐NMR‐Spektren mit variablen Sättigungszeiten (0.5–2.0 s) aufgenommen.…”

Section: Methodsunclassified

Molekulare Erkennung von UDP-Gal durchβ-1,4-Galactosyltransferase T1

Biet

Peters

2001

Angew. Chem.

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Mit STD‐NMR‐Untersuchungen kann das Bindungsepitop von UDP‐Gal und UDP‐Glc im Komplex mit der Glycosyltransferase β4Gal‐T1 (siehe Abbildung) entschlüsselt werden. Während das Enzym die Galactoseeinheit der UDP‐Gal erkennt, ist keinerlei Kontakt der Glucoseeinheit der UDP‐Glc mit der Proteinoberfläche zu beobachten. Dies erklärt, dass UDP‐Glc zwar von β4Gal‐T1 gebunden wird, aber keine Übertragungsreaktion der Glucose auf einen Acceptor stattfindet.

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“…Yet in ruptured vesicles, or on solubilization, the enzyme requires 50-100 times as much. The interpretation of this remains uncertain (Kuhn et al, 1991). At site 2 a quite different spectrum of cations can activate.…”

Section: Cation Binding By Lactose Synthetasementioning

confidence: 98%