Golgi targeting of Drosophila melanogaster beta4GalNAcTB requires a DHHC protein family-related protein as a pilot.
Drosophila melanogaster beta4GalNAcTB mutant flies revealed that this particular N-acetylgalactosaminyltransferase is predominant in the formation of lacdiNAc (GalNAcbeta1,4GlcNAc)-modified glycolipids, but enzymatic activity could not be confirmed for the cloned enzyme. Using a heterologous expression cloning approach, we isolated beta4GalNAcTB together with beta4GalNAcTB pilot (GABPI), a multimembrane-spanning protein related to Asp-His-His-Cys (DHHC) proteins but lacking the DHHC consensus sequence. In the absence of GABPI, inactive beta4GalNAcTB is trapped in the endoplasmic reticulum (ER). Coexpression of beta4GalNAcTB and GABPI generates the active enzyme that is localized together with GABPI in the Golgi. GABPI associates with beta4GalNAcTB and, when expressed with an ER retention signal, holds active beta4GalNAcTB in the ER. Importantly, treatment of isolated membrane vesicles with Triton X-100 disturbs beta4GalNAcTB activity. This phenomenon occurs with multimembrane-spanning glycosyltransferases but is normally not a property of glycosyltransferases with one membrane anchor. In summary, our data provide evidence that GABPI is required for ER export and activity of beta4GalNAcTB.
Johswich
Anita
A
Department of Cellular Chemistry, Hannover Medical School, Hannover, Germany.
Kraft
Benjamin
B
Wuhrer
Manfred
M
Berger
Monika
M
Deelder
André M
AM
Hokke
Cornelis H
CH
Gerardy-Schahn
Rita
R
Bakker
Hans
H
D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D029721:Drosophila Proteins / Q000737:chemistry / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D015723:Gene Library; D006017:Glycolipids / Q000378:metabolism; D006031:Glycosylation; D006056:Golgi Apparatus / Q000378:metabolism*; D006801:Humans; D007785:Lactose / Q000031:analogs & derivatives / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D017350:N-Acetylgalactosaminyltransferases / Q000737:chemistry / Q000378:metabolism*; D017830:Octoxynol / Q000494:pharmacology; D025941:Protein Interaction Mapping; D021381:Protein Transport; D012503:Saponins / Q000494:pharmacology
D029721:Drosophila Proteins; C535068:GABPI protein, Drosophila; D006017:Glycolipids; D008565:Membrane Proteins; D012503:Saponins; C093701:N-acetylgalactosaminyl-1-4-N-acetylglucosamine; D017830:Octoxynol; D017350:N-Acetylgalactosaminyltransferases; C535067:beta4GalNAcTB protein, Drosophila; C023220:(N-acetylneuraminyl)-galactosylglucosylceramide N-acetylgalactosaminyltransferase; D007785:Lactose