Golgi GDP-mannose Uptake Requires Leishmania LPG2

doi:10.1074/jbc.272.6.3799

Golgi GDP-mannose Uptake Requires Leishmania LPG2

A MEMBER OF A EUKARYOTIC FAMILY OF PUTATIVE NUCLEOTIDE-SUGAR TRANSPORTERS*

^‡ Department of Biochemistry, University of Kentucky Medical Center, Lexington, Kentucky 40536
^¶ Department of Molecular Microbiology and Molecular Genetics, Washington University School of Medicine, St. Louis, Missouri 63110, and the
** Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02130

^‡‡ To whom correspondence should be addressed:
Dept. of Biochemistry, University of Kentucky Medical Center, Lexington, KY 40536.
Tel.: 606-323-6693; Fax: 606-323-1037; E-mail: turco{at}pop.uky.edu

Abstract

The synthesis of glycoconjugates within the secretory pathway of eukaryotes requires the provision of lumenal nucleotide-sugar substrates. This is particularly important for eukaryotic microbes such as Leishmania because they must synthesize considerable amounts of extracellular and cell surface glycoconjugates that play significant roles in the infectious cycle. Here we used properly oriented sealed microsomes to characterize lumenal uptake of GDP-Man in Leishmania donovani In this system, GDP-Man uptake was saturable with an apparent K_m for GDP-Man of 0.3 μM and facilitated its use as a donor substrate for lipophosphoglycan (LPG) synthesis. A lpg2⁻ deletion mutant showed loss of GDP-Man but not UDP-Gal uptake, which was restored by introduction of the gene LPG2 Immunoelectron microscopy localized an active, epitope-tagged LPG2 protein to the Golgi apparatus. Thus, LPG2 is required for nucleotide-sugar transport activity and probably encodes this Golgi transporter. LPG2 belongs to a large family of eukaryotic genes that potentially encode transporters with different substrate specificities and/or cellular locations. In the future, the amenability of the Leishmania system to biochemical and genetic manipulation will assist in functional characterization of nucleotide-sugar transports from this and other eukaryotes. Furthermore, since LPG2 plays an important role in the Leishmania infectious cycle and mammalian cells lack a Golgi GDP-Man transporter, this activity may offer a new target for chemotherapy.

Footnotes

↵^§ Recipient of a University of Kentucky predoctoral fellowship.
↵^| Burroughs Wellcome Scholar in Molecular Parasitology.
↵* This work was funded in part by National Institutes of Health Grant AI31078. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

NST

nucleotide-sugar transporter

LPG

lipophosphoglycan

EST

expressed sequence tag

DIDS

4,4-diisothiocyanatostilbene-2,2′-disulfonic acid

TLCK

N^α-p-tosyl-L-lysine chloromethyl ketone

HA

hemagglutinin.
↵²N. Dean, D. Ma, S. Beverley, and S. Turco, unpublished observations.
- Received August 15, 1996.
- Revision received October 11, 1996.