The signal for Golgi retention of bovine β1,4-galactosyltransferase is in the transmembrane domain

Teasdale, Rohan D., Dagostaro, Giacomo and Gleeson, Paul A. (1992) The signal for Golgi retention of bovine β1,4-galactosyltransferase is in the transmembrane domain. Journal of Biological Chemistry, 267 6: 4084-4096.

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Author Teasdale, Rohan D.
Dagostaro, Giacomo
Gleeson, Paul A.
Title The signal for Golgi retention of bovine β1,4-galactosyltransferase is in the transmembrane domain
Journal name Journal of Biological Chemistry   Check publisher's open access policy
ISSN 0021-9258
1083-351X
Publication date 1992-02-25
Sub-type Article (original research)
Open Access Status File (Publisher version)
Volume 267
Issue 6
Start page 4084
End page 4096
Total pages 13
Place of publication Bethesda, MD, United States
Publisher American Society for Biochemistry and Molecular Biology
Language eng
Formatted abstract
The expression and localization of bovine β1,4-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 Nin/Cout 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.
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status Non-UQ

Document type: Journal Article
Sub-type: Article (original research)
Collection: Institute for Molecular Bioscience - Publications
 
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