Calsenilin and CALP interact with the cytoplasmic tail of UDP-Gal:GA2/GM2/GD2 beta-1,3-galactosyltransferase.
GalT2 (UDP-Gal:GA2/GM2/GD2 beta-1,3-galactosyltransferase) is a Golgi-resident type II membrane protein that participates in the synthesis of glycosphingolipids. The molecular determinants for traffic and localization of this and other glycosyltransferases are still poorly characterized. Considering the possibility that interactions with other proteins may influence these processes, in the present study we carried out a yeast two-hybrid screening using elements of the N-terminal domain of GalT2 as bait. In this screening, we identified calsenilin and its close homologue CALP (calsenilin-like protein), both members of the recoverin-NCS (neuronal calcium sensor) family of calcium-binding proteins. In vitro, GalT2 binds to immobilized recombinant CALP, and CALP binds to immobilized peptides with the GalT2 cytoplasmic tail sequence. GalT2 and calsenilin interact physically when co-expressed in CHO (Chinese-hamster ovary)-K1 cells. The expression of CALP or calsenilin affect Golgi localization of GalT2, and of two other glycosyltransferases, SialT2 (CMP-NeuAc:GM3 sialyltransferase) and GalNAcT (UDP-GalNAc:lactosylceramide/GM3/GD3 beta1-4 N-acetylgalactosaminyltransferase), by redistributing them from the Golgi to the ER (endoplasmic reticulum), whereas the localization of the VSV-G (G-protein of the vesicular stomatitis virus) or the Golgin GM130 was essentially unaffected. Conversely, the expression of GalT2 affects the localization of calsenilin and CALP by shifting a fraction of the molecules from being mostly diffuse in the cytosol, to clustered structures in the perinuclear region. These combined in vivo and in vitro results suggest that CALP and calsenilin are involved in the trafficking of Golgi glycosyltransferases.
Quintero
Cristián A
CA
Centro de Investigaciones en Química Biológica de Córdoba, CIQUIBIC (UNC-CONICET), Departamento de Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Ciudad Universitaria, X5000HUA Córdoba, Argentina.
Valdez-Taubas
Javier
J
Ferrari
Mariana L
ML
Haedo
Sergio D
SD
Maccioni
Hugo J F
HJ
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005700:Galactosyltransferases / Q000737:chemistry / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D051605:Kv Channel-Interacting Proteins / Q000378:metabolism* / Q000502:physiology; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution; D020798:Two-Hybrid System Techniques
D051605:Kv Channel-Interacting Proteins; C108454:GD2 beta1,3-galactosyltransferase; D005700:Galactosyltransferases