Interaction in vitro; enzyme produced as a recombinant fusion protein in bacterial system; enzyme target produced as a recombinant fusion protein in bacterial system.
Assayed by immunocytochemical staining against LacdiNAc, a product of the β4GalNAcTB glycosyltransferase reaction, on the cell surface. Both GABPI and β4GalNAcTB cDNAs were required.
Source was HEK293 (human embryonic kidney) cell line transfected with tagged GABPI and β4GalNAcTB constructs.
Source was S2 cell line transfected with tagged GABPI and β4GalNAcTB constructs.
Assayed for localization of β4GalNAcTB protein to the Golgi. Specific regions of β4GalNAcTB protein analyzed by creating chimeric proteins with β4GalNAcTA, which does not interact with the GABPI protein.
Transiently transfected HEK293 (human embryonic kidney) cells, contransfected with β4GalNAcTB and HA-insertion constructs of GABPI.
Insertion of HA tags in the predicted luminal loops of the GABPI protein disrupted the reaction.
Ability of GABPI protein to activate β4GalNAcTB protein on the cell surface of transfected HEK293 cells assayed by immunostaining against LacdiNAc, a product of the β4GalNAcTB glycosyltransferase reaction.
Within TMD3/4 luminal loop; replacement of P176 with Ala eliminates binding.
Within TMD3/4 luminal loop; replacement of both E178 and E178 with Ala eliminates binding (replacement of one or the other not sufficient).
Within TMD5/6 luminal loop; replacement of H335 with Ala eliminates binding.
Within TMD5/6 luminal loop; replacement of P346 with Ala eliminates binding.
Within TMD5/6 luminal loop; replacement of both L348 and L349 with Ala eliminates binding (replacement of one or the other not sufficient).
Within TMD5/6 luminal loop; replacement of C353 with Ala eliminates binding.
Transiently transfected HEK293 (human embryonic kidney) cells, contransfected with β4GalNAcTB and GABPI constructs.
Ability of GABPI protein to activate β4GalNAcTB protein on the cell surface of transfected HEK293 cells assayed by immunostaining against LacdiNAc, a product of the β4GalNAcTB glycosyltransferase reaction.
Transiently transfected HEK293 (human embryonic kidney) cells, transfected with β4GalNAcTB-GABPI fusion constructs.
Expressing mutant GABPI and wild-type β4GalNAcTB as a fusion protein overcomes disruption of the interaction seen with some amino acid replacements (H335A and P346A) in the TMD5/6 luminal loop of the GABPI protein.
Ability of GABPI protein to activate β4GalNAcTB protein on the cell surface of transfected HEK293 cells assayed by immunostaining against LacdiNAc, a product of the β4GalNAcTB glycosyltransferase reaction.
Replacement of both Y38 and Y40 with Ser eliminates binding (replacement of one or the other not sufficient).
Transiently transfected HEK293 (human embryonic kidney) cells, contransfected with β4GalNAcTB and GABPI constructs.
Ability of GABPI protein to activate β4GalNAcTB protein on the cell surface of transfected HEK293 cells assayed by immunostaining against LacdiNAc, a product of the β4GalNAcTB glycosyltransferase reaction.
