A Non-Golgi α1,2-Fucosyltransferase That Modifies Skp1 in the Cytoplasm of Dictyostelium

Abstract

Skp1 is a subunit of the SCF-E3 ubiquitin ligase that targets cell cycle and other regulatory factors for degradation. In Dictyostelium, Skp1 is modified by a pentasaccharide containing the type 1 blood group H trisaccharide at its core. To address how the third sugar, fucose ␣1,2-linked to galactose, is attached, a proteomics strategy was applied to determine the primary structure of FT85, previously shown to copurify with the GDPFuc:Skp1 ␣1,2-fucosyltransferase. Tryptic-generated peptides of FT85 were sequenced de novo using Q-TOF tandem mass spectrometry. Degenerate primers were used to amplify FT85 genomic DNA, which was further extended by a novel linker polymerase chain reaction method to yield an intronless open reading frame of 768 amino acids. Disruption of the FT85 gene by homologous recombination resulted in viable cells, which had altered light scattering properties as revealed by flow cytometry. FT85 was necessary and sufficient for Skp1 fucosylation, based on biochemical analysis of FT85 mutant cells and Escherichia coli that express FT85 recombinantly. FT85 lacks sequence motifs that characterize all other known ␣1,2-fucosyltransferases and lacks the signal-anchor sequence that targets them to the secretory pathway. The C-terminal region of FT85 harbors motifs found in inverting Family 2 glycosyltransferase domains, and its expression in FT85 mutant cells restores fucosyltransferase activity toward a simple disaccharide substrate. Whereas most prokaryote and eukaryote Family 2 glycosyltransferases are membranebound and oriented toward the cytoplasm where they glycosylate lipid-linked or polysaccharide precursors prior to membrane translocation, the soluble, eukaryotic Skp1-fucosyltransferase modifies a protein that resides in the cytoplasm and nucleus.

PpATG9 Encodes a Novel Membrane Protein That Traffics to Vacuolar Membranes, Which Sequester Peroxisomes during Pexophagy inPichia pastoris

“…The null mutant of PpVps15 was provided by Dr. J. Cregg (Keck Graduate Institute; Stasyk et al, 1999). PpVAC8 was cloned from genomic DNA using degenerate primers combined with linker-mediated PCR (van Der Wel et al, 2001). The entire gene was sequenced and assembled (NCBI accession number AY886543).…”

“…The site of insertion of the pREMI-Z and identification of the disrupted gene was done as described . Based on the genomic sequences around the pREMI-Z insertion site that was isolated along with the pREMI-Z vector from the R19 mutant, GSA14 was cloned and sequenced from genomic DNA using a linker-mediated PCR method previously described (van Der Wel et al, 2001). We were able to completely assemble the GSA14 gene (National Center for Biotechnology Information [NCBI] accession number AY075105) and show it encodes a protein homologous to ScAtg9 of S. cerevisiae, using the adapted ATG nomenclature for those genes uniquely essential for autophagy .…”

“De novo” peptide sequencing by MALDI-quadrupole-ion trap mass spectrometry: A preliminary study

“…Notable in this regard is the use of 18 O incorporation at the carboxyl-termini of peptides during protein hydrolysis [14 -17]. For underivatized peptides, ESI has found use for de novo sequencing under a variety of conditions, which include collision-induced dissociation (CID) in the triple quadrupole mass analyzer [18 -20] and the QqTOF mass analyzer [18,19,[21][22][23][24][25][26], resonant excitation in the quadrupole ion trap mass analyzer [14,[27][28][29], and electron capture dissociation in the Fourier transform ion cyclotron resonance mass analyzer [30 -34]. MALDI de novo sequencing has been carried out using CID and metastable decay in single- [35] and double-stage [36 -39] TOF instruments and with CID in QqTOF instruments [40 -44].…”

Role of SP65 in Assembly of the Dictyostelium discoideum Spore Coat

“…The previously reported peptide sequence VRGNPT CLRNHDGI (38) was found to be encoded perfectly by a 309-nucleotide (nt) sequence present in a database of random sequences of shotgun-cloned Dictyostelium genomic DNA (gDNA) available at the time. Primers derived from this nucleotide sequence were used to clone additional gDNA (unpublished data) using inverse PCR and linker-mediated PCR (20,27,28). Segments of the new sequences matched other gDNA sequences in the databases.…”