Mutational analysis of the Golgi retention signal of bovine beta-1,4-galactosyltransferase

Masibay, Arni S.; Balaji, Petety V.; Boeggeman, Elisabeth; Qasba, Pradman K.

doi:10.1016/s0021-9258(18)98431-0

Cited by 126 publications

(21 citation statements)

References 57 publications

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“…Alternatively, a cluster of Gb 3 molecules might tend to segregate in membrane bilayers of a particular thickness. This could play a role in intracellular trafficking of the toxin; different organelles have different lipid compositions, and the length of membrane-spanning helixes in membrane proteins has been shown to influence their subcellular distribution ( , ).…”

Section: Resultsmentioning

confidence: 99%

Structure of the Shiga-like Toxin I B-Pentamer Complexed with an Analogue of Its Receptor Gb₃^,

et al. 1998

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Shiga-like toxin I (SLT-I) is a virulence factor of Escherichia coli strains that cause disease in humans. Like other members of the Shiga toxin family, it consists of an enzymatic (A) subunit and five copies of a binding subunit (the B-pentamer). The B-pentamer binds to a specific glycolipid, globotriaosylceramide (Gb3), on the surface of target cells and thereby plays a crucial role in the entry of the toxin. Here we present the crystal structure at 2.8 A resolution of the SLT-I B-pentamer complexed with an analogue of the Gb3 trisaccharide. The structure reveals a surprising density of binding sites, with three trisaccharide molecules bound to each B-subunit monomer of 69 residues. All 15 trisaccharides bind to one side of the B-pentamer, providing further evidence that this side faces the cell membrane. The structural model is consistent with data from site-directed mutagenesis and binding of carbohydrate analogues, and allows the rational design of therapeutic Gb3 analogues that block the attachment of toxin to cells.

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Section: Resultsmentioning

confidence: 99%

Structure of the Shiga-like Toxin I B-Pentamer Complexed with an Analogue of Its Receptor Gb₃^,

et al. 1998

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“…However, a longer stretch of 23 Leu residues did not provide an efficient retention signal (7). Similarly, a 4-residue insertion into the transmembrane domain of galactosyltransferase reduced its retention in the Golgi (11). The reverse effect has been shown with the influenza virus neuraminidase which shifted from the plasma membrane to the Golgi and the ER when the number of residues in the trans-membrane domain was reduced (12).…”

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confidence: 99%

“…One possible model is retention by preferential interaction with membranes of optimal thickness (1). Both Bretscher and Munro (10) and Masibay et al (11) have shown that trans-membrane domains of Golgi proteins are shorter (average 15 residues) than trans-membrane domains of plasma membrane proteins (average 20 residues). It has therefore been suggested that, if membrane proteins with short trans-membrane domains interact better with thin lipid bilayers and if the lipid bilayer in the Golgi is thinner than that in the plasma membrane, then this could be the mechanism of retention in the Golgi (10,11).…”

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confidence: 99%

Hydrophobic Mismatch and the Incorporation of Peptides into Lipid Bilayers: A Possible Mechanism for Retention in the Golgi

et al. 1998

Biochemistry

129

137

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Preferential interaction of trans-membrane alpha-helices whose hydrophobic length matches the hydrophobic thickness of the lipid bilayer could be a mechanism of retention in the Golgi apparatus. We have used fluorescence methods to study the interaction of peptides Ac-K2-G-Lm-W-Ln-K2-A-amide (Pm+n) with bilayers of phosphatidylcholines with chain lengths between C14 and C24. The peptide P22 (m = 10, n = 12) incorporates into all bilayers, but P16 (m = 7, n = 9) does not incorporate into bilayers when the fatty acyl chain length is C24 and only partly incorporates into bilayers where the chain length is C22. The strongest binding is seen when the hydrophobic length of the peptide matches the calculated hydrophobic thickness of the bilayer. It is suggested that a too-thin bilayer can match to a too-long peptide both by stretching of the lipid and by tilting of the peptide. However, a too-thick bilayer can only match a too-thin peptide by compression of the lipid, which becomes energetically unfavorable when the difference between the bilayer thickness and the peptide length exceeds about 10 A. The presence of cholesterol in the bilayer leads to a marked reduction in the incorporation of P16 into bilayers where the chain length is C18. Hydrophobic mismatch could explain retention of proteins with short trans-membrane alpha-helical domains in the Golgi, the effect following largely from the low concentration of cholesterol in the Golgi membrane compared to that in the plasma membrane.

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“…Much effort has been directed towards identifying the "Golgi retention signal" of early type II proteins. Many studies indicate that the transmembrane domain is critical (2,14,32,39,57,89,98,99,111) with some researchers finding that the adjacent regions are also involved (15,19,58,59,62,66,83,100,104). However, several groups have argued that retention is conferred by multiple domains of the molecule (3,7,12,32,54,90).…”

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confidence: 99%

Localization of a yeast early Golgi mannosyltransferase, Och1p, involves retrograde transport.

Harris

Waters

1996

The Journal of Cell Biology

141

122

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To analyze the mechanism of integral membrane protein localization in the early Golgi apparatus of Saccharomyces cerevisiae, we have used Och1p, a cis-Golgi mannosyltransferase. A series of influenza virus hemagglutinin (HA) epitope-tagged fusion proteins was constructed in which invertase is appended to the Golgi-luminal carboxy terminus of full-length Och1p. Several constructs included a Kex2p cleavage site between the Och1p and invertase moieties to monitor transit to the Kex2p-containing TGN. Cells expressing an Och1p-invertase fusion do not secrete invertase, but those expressing an Och1p-Kex2p site-invertase fusion protein secrete high levels of invertase in a Kex2p-dependent manner. The Och1p-Kex2p site-invertase fusion protein is cleaved with a half-time of 5 min, and the process proceeds to completion. Before cleavage the protein receives glycosyl modifications indicative of passage through the medial- and trans-Golgi, therefore cleavage occurs after ordered anterograde transport through the Golgi to the TGN. Transit to distal compartments is not induced by the invertase moiety, since noninvertase fusion constructs encounter the same glycosyltransferases and Kex2p as well. The Och1p-HA moiety, irrespective of whether it is generated by cleavage of the fusion protein in the TGN or synthesized de novo, is degraded with a half-time of about 60 min. Thus, the half-time of degradation is 12-fold longer than the time required to reach the TGN. At steady state, de novo- synthesized and TGN-generated HA epitope-tagged Och1p reside in a compartment with a buoyant density identical to that of wild-type Och1p and distinct from that of the vacuole or the TGN. Finally, och1 null cells that express an Ochlp fusion construct known to rapidly encounter the TGN glycosylate invertase to the same extent as wild-type cells, indicating that they have phenotypically wild-type Och1p activity. These results lead us to propose a model for Och1p-HA localization that involves movement to distal compartments, at least as far as the TGN, followed by retrieval to the cis compartment, presumably by vesicular transport.

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Mutational analysis of the Golgi retention signal of bovine beta-1,4-galactosyltransferase

Cited by 126 publications

References 57 publications

Structure of the Shiga-like Toxin I B-Pentamer Complexed with an Analogue of Its Receptor Gb₃^,

Structure of the Shiga-like Toxin I B-Pentamer Complexed with an Analogue of Its Receptor Gb₃^,

Hydrophobic Mismatch and the Incorporation of Peptides into Lipid Bilayers: A Possible Mechanism for Retention in the Golgi

Localization of a yeast early Golgi mannosyltransferase, Och1p, involves retrograde transport.

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Mutational analysis of the Golgi retention signal of bovine beta-1,4-galactosyltransferase

Cited by 126 publications

References 57 publications

Structure of the Shiga-like Toxin I B-Pentamer Complexed with an Analogue of Its Receptor Gb3,

Structure of the Shiga-like Toxin I B-Pentamer Complexed with an Analogue of Its Receptor Gb3,

Hydrophobic Mismatch and the Incorporation of Peptides into Lipid Bilayers: A Possible Mechanism for Retention in the Golgi

Localization of a yeast early Golgi mannosyltransferase, Och1p, involves retrograde transport.

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Product

Resources

About

Structure of the Shiga-like Toxin I B-Pentamer Complexed with an Analogue of Its Receptor Gb₃^,

Structure of the Shiga-like Toxin I B-Pentamer Complexed with an Analogue of Its Receptor Gb₃^,