title: Time-resolved fluorescence imaging reveals differential interactions of N-glycan processing enzymes across the Golgi stack in planta.
abstract: N-Glycan processing is one of the most important cellular protein modifications in plants and as such is essential for plant development and defense mechanisms. The accuracy of Golgi-located processing steps is governed by the strict intra-Golgi localization of sequentially acting glycosidases and glycosyltransferases. Their differential distribution goes hand in hand with the compartmentalization of the Golgi stack into cis-, medial-, and trans-cisternae, which separate early from late processing steps. The mechanisms that direct differential enzyme concentration are still unknown, but the formation of multienzyme complexes is considered a feasible Golgi protein localization strategy. In this study, we used two-photon excitation-Förster resonance energy transfer-fluorescence lifetime imaging microscopy to determine the interaction of N-glycan processing enzymes with differential intra-Golgi locations. Following the coexpression of fluorescent protein-tagged amino-terminal Golgi-targeting sequences (cytoplasmic-transmembrane-stem [CTS] region) of enzyme pairs in leaves of tobacco (Nicotiana spp.), we observed that all tested cis- and medial-Golgi enzymes, namely Arabidopsis (Arabidopsis thaliana) Golgi α-mannosidase I, Nicotiana tabacum β1,2-N-acetylglucosaminyltransferase I, Arabidopsis Golgi α-mannosidase II (GMII), and Arabidopsis β1,2-xylosyltransferase, form homodimers and heterodimers, whereas among the late-acting enzymes Arabidopsis β1,3-galactosyltransferase1 (GALT1), Arabidopsis α1,4-fucosyltransferase, and Rattus norvegicus α2,6-sialyltransferase (a nonplant Golgi marker), only GALT1 and medial-Golgi GMII were found to form a heterodimer. Furthermore, the efficiency of energy transfer indicating the formation of interactions decreased considerably in a cis-to-trans fashion. The comparative fluorescence lifetime imaging of several full-length cis- and medial-Golgi enzymes and their respective catalytic domain-deleted CTS clones further suggested that the formation of protein-protein interactions can occur through their amino-terminal CTS region.
authors: Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, Austria.
authors: Jennifer
authors: J
authors: Schoberer
authors: Eva
authors: E
authors: Liebminger
authors: Stanley W
authors: SW
authors: Botchway
authors: Richard
authors: R
authors: Strasser
authors: Chris
authors: C
authors: Hawes
mesh_terms: D000818:Animals; D017360:Arabidopsis / Q000201:enzymology*; D015415:Biomarkers / Q000378:metabolism; D020134:Catalytic Domain; D031541:Fluorescence Resonance Energy Transfer; D016695:Glycosyltransferases / Q000378:metabolism*; D006056:Golgi Apparatus / Q000201:enzymology*; D047468:Immunoprecipitation; D008361:Mannosidases / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000379:methods*; D008969:Molecular Sequence Data; D017785:Photons; D059828:Plant Cells / Q000201:enzymology; D018515:Plant Leaves / Q000201:enzymology; D011134:Polysaccharides / Q000378:metabolism*; D011485:Protein Binding; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013347:Subcellular Fractions / Q000201:enzymology; D013997:Time Factors; D014026:Nicotiana / Q000201:enzymology*
chemical_list: D015415:Biomarkers; D011134:Polysaccharides; D011993:Recombinant Fusion Proteins; D016695:Glycosyltransferases; D008361:Mannosidases
references: J Biosci Bioeng. 2012 Jan;113(1):48-54
references: 22024534
references: J Biol Chem. 2001 Aug 3;276(31):28641-9
references: 11356854
references: Plant Mol Biol. 2002 Sep;50(2):273-81
references: 12175019
references: FEBS Lett. 2000 Apr 21;472(1):105-8
references: 10781814
references: Plant Cell. 2006 Nov;18(11):3182-200
references: 17138701
references: Plant Physiol. 2007 Oct;145(2):339-50
references: 17693538
references: J Biol Chem. 2010 Jun 4;285(23):17771-7
references: 20378551
references: Proc Natl Acad Sci U S A. 2011 Dec 13;108(50):20225-30
references: 22135470
references: Cell Microbiol. 2010 Jun;12(6):716-24
references: 20408850
references: J Cell Sci. 2006 Dec 15;119(Pt 24):5087-97
references: 17158913
references: Traffic. 2009 Aug;10(8):1034-46
references: 19490533
references: Plant Mol Biol. 2005 May;58(1):109-22
references: 16028120
references: J Biol Chem. 2005 Feb 18;280(7):5423-9
references: 15582997
references: Plant J. 2003 Jan;33(1):189-203
references: 12943552
references: Curr Opin Plant Biol. 2004 Apr;7(2):171-81
references: 15003218
references: J Biomed Opt. 2003 Jul;8(3):362-7
references: 12880340
references: J Biol Chem. 2011 Nov 4;286(44):38329-38340
references: 21911486
references: Plant Cell. 2010 Apr;22(4):1333-43
references: 20424177
references: Plant Physiol. 2010 Sep;154(1):78-97
references: 20631319
references: J Membr Biol. 2004 Mar 1;198(1):1-13
references: 15209092
references: FEBS Lett. 1993 Sep 6;330(1):1-4
references: 8370450
references: Biochem Cell Biol. 1986 Mar;64(3):163-81
references: 3521675
references: Biophys J. 2010 Aug 9;99(4):1274-83
references: 20713013
references: BMC Cell Biol. 2005 Apr 26;6(1):22
references: 15854225
references: Proc Natl Acad Sci U S A. 2001 Feb 13;98(4):1625-30
references: 11172001
references: Planta. 2012 Jul;236(1):115-28
references: 22270560
references: J Cell Sci. 1996 Jul;109 ( Pt 7):1975-89
references: 8832420
references: Nat Protoc. 2011 Aug 11;6(9):1324-40
references: 21886099
references: J Cell Physiol. 2009 Jul;220(1):144-54
references: 19277980
references: J Biomol Screen. 1999;4(6):355-362
references: 10838432
references: Proc Natl Acad Sci U S A. 2000 Jan 18;97(2):668-73
references: 10639137
references: Plant Cell. 2007 Jul;19(7):2278-92
references: 17630273
references: J Cell Biol. 1995 Jun;129(6):1543-58
references: 7790353
references: Glycobiology. 1997 Feb;7(1):1-13
references: 9061359
references: Plant Cell. 2002 Jun;14(6):1293-309
references: 12084828
references: Plant Cell. 2010 Sep;22(9):2930-42
references: 20876830
references: FEBS Lett. 1999 Jun 18;453(1-2):169-73
references: 10403396
references: Proc Natl Acad Sci U S A. 2005 Feb 22;102(8):3135-40
references: 15703292
references: Trends Cell Biol. 1991 Dec;1(6):141-4
references: 14731855
references: Plant Methods. 2012 Jul 12;8(1):25
references: 22789293
references: J Biol Chem. 1996 Mar 29;271(13):7758-66
references: 8631818
references: Plant Cell. 2003 Oct;15(10):2273-84
references: 12972670
references: EMBO J. 1994 Feb 1;13(3):562-74
references: 8313901
references: Plant J. 2006 Mar;45(5):789-803
references: 16460512
references: Biochimie. 2001 Aug;83(8):763-73
references: 11530209
references: Plant Mol Biol. 1998 Sep;38(1-2):31-48
references: 9738959
references: Plant Cell. 2009 Dec;21(12):3850-67
references: 20023195
references: Proc Natl Acad Sci U S A. 2001 Nov 6;98(23):12984-9
references: 11687650
references: Plant Physiol. 2012 Aug;159(4):1355-66
references: 22665445
references: FEBS Lett. 2011 Jul 21;585(14):2193-8
references: 21669202
references: Traffic. 2010 Nov;11(11):1429-44
references: 20716110
references: Plant J. 1998 Aug;15(3):441-7
references: 9750355
references: Plant Mol Biol. 2012 May;79(1-2):21-33
references: 22328055
references: Plant Physiol. 1999 Dec;121(4):1127-42
references: 10594100
references: Mol Plant. 2011 Mar;4(2):220-8
references: 21307368
references: J Biol Chem. 2002 Nov 22;277(47):44801-8
references: 12235155
references: Proc Natl Acad Sci U S A. 2008 Apr 15;105(15):5933-8
references: 18408158
references: Nat Protoc. 2006;1(4):2019-25
references: 17487191
references: Curr Biol. 1999 Mar 11;9(5):269-72
references: 10074454
references: Neurochem Res. 2012 Jun;37(6):1325-34
references: 22388569
references: Trends Cell Biol. 1999 Feb;9(2):48-52
references: 10087617
references: Traffic. 2009 Jan;10(1):101-15
references: 18939950