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Journal of Bacteriology, December 2008, p. 8045-8052, Vol. 190, No. 24
0021-9193/08/$08.00+0     doi:10.1128/JB.01200-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Defining the Topology of the N-Glycosylation Pathway in the Halophilic Archaeon Haloferax volcanii{triangledown}

Noa Plavner and Jerry Eichler*

Department of Life Sciences, Ben Gurion University, Beersheva 84105, Israel

Received 26 August 2008/ Accepted 2 October 2008

In Eukarya, N glycosylation involves the actions of enzymes working on both faces of the endoplasmic reticulum membrane. The steps of bacterial N glycosylation, in contrast, transpire essentially on the cytoplasmic side of the plasma membrane, with only transfer of the assembled glycan to the target protein occurring on the external surface of the cell. For Archaea, virtually nothing is known about the topology of enzymes involved in assembling those glycans that are subsequently N linked to target proteins on the external surface of the cell. To remedy this situation, subcellular localization and topology predictive algorithms, protease accessibility, and immunoblotting, together with cysteine modification following site-directed mutagenesis, were enlisted to define the topology of Haloferax volcanii proteins experimentally proven to participate in the N-glycosylation process. AglJ and AglD, involved in the earliest and latest stages, respectively, of assembly of the pentasaccharide decorating the H. volcanii S-layer glycoprotein, were shown to present their soluble N-terminal domain, likely containing the putative catalytic site of each enzyme, to the cytosol. The same holds true for Alg5-B, Dpm1-A, and Mpg1-D, proteins putatively involved in this posttranslational event. The results thus point to the assembly of the pentasaccharide linked to certain Asn residues of the H. volcanii S-layer glycoprotein as occurring within the cell.

* Corresponding author. Mailing address: Dept. of Life Sciences, Ben Gurion University, P.O. Box 653, Beersheva 84105, Israel. Phone: 972 8646 1343. Fax: 972 8647 9175. E-mail: jeichler{at}bgu.ac.il

{triangledown} Published ahead of print on 17 October 2008.

Journal of Bacteriology, December 2008, p. 8045-8052, Vol. 190, No. 24
0021-9193/08/$08.00+0     doi:10.1128/JB.01200-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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