Identification of a site in Sar1 involved in the interaction with the cytoplasmic tail of glycolipid glycosyltransferases.
Glycolipid glycosyltransferases (GGT) are transported from the endoplasmic reticulum (ER) to the Golgi, their site of residence, via COPII vesicles. An interaction of a (R/K)X(R/K) motif at their cytoplasmic tail (CT) with Sar1 is critical for the selective concentration in the transport vesicles. In this work using computational docking, we identify three putative binding pockets in Sar1 (sites A, B, and C) involved in the interaction with the (R/K)X(R/K) motif. Sar1 mutants with alanine replacement of amino acids in site A were tested in vitro and in cells. In vitro, mutant versions showed a reduced ability to bind immobilized peptides with the CT sequence of GalT2. In cells, Sar1 mutants (Sar1(D198A)) specifically affect the exiting of GGT from the ER, resulting in an ER/Golgi concentration ratio favoring the ER. Neither the typical Golgi localization of GM130 nor the exiting and transport of the G protein of the vesicular stomatitis virus were affected. The protein kinase inhibitor H89 produced accumulation of Sec23, Sar1, and GalT2 at the ER exit sites; Sar1(D189A) also accumulated at these sites, but in this case GalT2 remained disperse along ER membranes. The results indicate that amino acids in site A of Sar1 are involved in the interaction with the CT of GGT for concentration at ER exiting sites.
Quintero
Cristián A
CA
Departamento de Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Ciudad Universitaria, Centro de Investigaciones en Química Biológica de Córdoba, X5000HUA Córdoba, Argentina.
Giraudo
Claudio G
CG
Villarreal
Marcos
M
Montich
Guillermo
G
Maccioni
Hugo J F
HJ
D020816:Amino Acid Motifs; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D022181:COP-Coated Vesicles / Q000201:enzymology / Q000235:genetics; D006224:Cricetinae; D003412:Cricetulus; D004721:Endoplasmic Reticulum / Q000201:enzymology* / Q000235:genetics; D005700:Galactosyltransferases / Q000235:genetics / Q000378:metabolism*; D006056:Golgi Apparatus / Q000201:enzymology* / Q000235:genetics; D007546:Isoquinolines / Q000494:pharmacology; D051379:Mice; D008958:Models, Molecular; D020559:Monomeric GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D011485:Protein Binding; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D013449:Sulfonamides / Q000494:pharmacology; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism
D007546:Isoquinolines; D047428:Protein Kinase Inhibitors; C079038:Sec23a protein, mouse; D013449:Sulfonamides; D033921:Vesicular Transport Proteins; D005700:Galactosyltransferases; C023235:glucosylceramide beta-1-4-galactosyltransferase; D020559:Monomeric GTP-Binding Proteins; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide