Glycobiology, 2001, Vol. 11, No. 4 283-295
© 2001 Oxford University Press
Analysis of Skp1 glycosylation and nuclear enrichment in Dictyostelium
Department of Anatomy and Cell Biology, Box 100235, 1600 SW Archer Road, University of Florida College of Medicine, Gainesville FL 32610-0235, USA
Skp1 is a subunit of SCF-E3 ubiquitin ligases and other protein complexes in the nucleus and cytoplasm of yeast and mammalian cells. In Dictyostelium, Skp1 is partially modified by an unusual pentasaccharide O-linked to hydroxyproline143. This modification was found to be susceptible to known prolyl hydroxylase inhibitors based on Mr-shift analysis using SDS–polyacrylamide gel electrophoresis/Western blotting. In addition, Dictyostelium Skp1 consists of 2 genetic isoforms, Skp1A and Skp1B, which differ by a single amino acid and appear to be expressed throughout the life cycle based on reverse-transcription polymerase chain reactions. The significance of these structural variations was examined by expressing myc-tagged Skp1s and mutants that lacked the glycosylation site. Gel-based Mr-shift studies showed that Skp1A and Skp1B are both nearly completely glycosylated during growth and early development, and mass spectrometry of glycopeptides showed that they were glycosylated similarly. Skp1 expressed later in prespore cells was not glycosylated, unlike bulk Skp1 persisting from earlier in development, but became glycosylated after return to growth medium. Skp1A and Skp1B were each concentrated in the nucleus and regions of the cytoplasm, based on immunofluorescence localization. However, when Skp1 glycosylation was blocked by mutation, prolyl hydroxylase inhibitors, or expression in prespore cells, nuclear concentration of Skp1 was not detected. Furthermore, nuclear concentration occurred in a mutant that attached only the core disaccharide to Skp1. Overall, there was no evidence for differential Skp1 isoform expression, glycosylation variants in the bulk Skp1 pool, or regulation of nuclear localization. However, these studies uncovered evidence that the glycosylation pathway is developmentally regulated and can function posttranslationally, and that core glycosylation is required for Skp1’s nuclear concentration.
1 To whom correspondence should be addressed
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  Z. A. Wang, H. van der Wel, Y. Vohra, T. Buskas, G.-J. Boons, and C. M. West Role of a Cytoplasmic Dual-function Glycosyltransferase in O2 Regulation of Development in Dictyostelium J. Biol. Chem., October 16, 2009; 284(42): 28896 - 28904. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. M. West, P. Nguyen, H. van der Wel, T. Metcalf, K. R. Sweeney, I. J. Blader, and G. W. Erdos Dependence of Stress Resistance on a Spore Coat Heteropolysaccharide in Dictyostelium Eukaryot. Cell, January 1, 2009; 8(1): 27 - 36. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. M. West, H. van der Wel, and Z. A. Wang Prolyl 4-hydroxylase-1 mediates O2 signaling during development of Dictyostelium Development, September 15, 2007; 134(18): 3349 - 3358. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. van der Wel, A. Ercan, and C. M. West The Skp1 Prolyl Hydroxylase from Dictyostelium Is Related to the Hypoxia-inducible Factor-{alpha} Class of Animal Prolyl 4-Hydroxylases J. Biol. Chem., April 15, 2005; 280(15): 14645 - 14655. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Ketcham, F. Wang, S. Z. Fisher, A. Ercan, H. van der Wel, R. D. Locke, k. Sirajud-Doulah, K. L. Matta, and C. M. West Specificity of a Soluble UDP-Galactose:Fucoside {alpha}1,3-Galactosyltransferase That Modifies the Cytoplasmic Glycoprotein Skp1 in Dictyostelium J. Biol. Chem., July 9, 2004; 279(28): 29050 - 29059. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Metcalf, K. Kelley, G. W. Erdos, L. Kaplan, and C. M. West Formation of the outer layer of the Dictyostelium spore coat depends on the inner-layer protein SP85/PsB Microbiology, February 1, 2003; 149(2): 305 - 317. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. van der Wel, H. R. Morris, M. Panico, T. Paxton, A. Dell, L. Kaplan, and C. M. West Molecular Cloning and Expression of a UDP-N-acetylglucosamine (GlcNAc):Hydroxyproline Polypeptide GlcNAc-transferase That Modifies Skp1 in the Cytoplasm of Dictyostelium J. Biol. Chem., November 22, 2002; 277(48): 46328 - 46337. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. van der Wel, S. Z. Fisher, and C. M. West A Bifunctional Diglycosyltransferase Forms the Fucalpha 1,2Galbeta 1,3-Disaccharide on Skp1 in the Cytoplasm of Dictyostelium J. Biol. Chem., November 22, 2002; 277(48): 46527 - 46534. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. M. West, P. Zhang, A. C. McGlynn, and L. Kaplan Outside-In Signaling of Cellulose Synthesis by a Spore Coat Protein in Dictyostelium Eukaryot. Cell, April 1, 2002; 1(2): 281 - 292. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. M. West, H. van der Wel, and E. A. Gaucher Complex glycosylation of Skp1 in Dictyostelium: implications for the modification of other eukaryotic cytoplasmic and nuclear proteins Glycobiology, February 1, 2002; 12(2): 17R - 27R. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. van der Wel, H. R. Morris, M. Panico, T. Paxton, S. J. North, A. Dell, J. M. Thomson, and C. M. West A Non-Golgi alpha 1,2-Fucosyltransferase That Modifies Skp1 in the Cytoplasm of Dictyostelium J. Biol. Chem., August 31, 2001; 276(36): 33952 - 33963. [Abstract] [Full Text] [PDF]
|
|