An insight into the orphan nucleotide sugar transporter SLC35A4.
SLC35A4 has been classified in the SLC35A subfamily based on amino acid sequence homology. Most of the proteins belonging to the SLC35 family act as transporters of nucleotide sugars. In this study, the subcellular localization of endogenous SLC35A4 was determined via immunofluorescence staining, and it was demonstrated that SLC35A4 localizes mainly to the Golgi apparatus. In silico topology prediction suggests that SLC35A4 has an uneven number of transmembrane domains and its N-terminus is directed towards the Golgi lumen. However, an experimental assay refuted this prediction: SLC35A4 has an even number of transmembrane regions with both termini facing the cytosol. In vivo interaction analysis using the FLIM-FRET approach revealed that SLC35A4 neither forms homomers nor associates with other members of the SLC35A subfamily except SLC35A5. Additional assays demonstrated that endogenous SLC35A4 is 10 to 40nm proximal to SLC35A2 and SLC35A3. To determine SLC35A4 function SLC35A4 knock-out cells were generated with the CRISPR-Cas9 approach. Although no significant changes in glycosylation were observed, the introduced mutation influenced the subcellular distribution of the SLC35A2/SLC35A3 complexes. Additional FLIM-FRET experiments revealed that overexpression of SLC35A4-BFP together with SLC35A3 and the SLC35A2-Golgi splice variant negatively affects the interaction between the two latter proteins. The results presented here strongly indicate a modulatory role for SLC35A4 in intracellular trafficking of SLC35A2/SLC35A3 complexes.
Laboratory of Biochemistry, Faculty of Biotechnology, University of Wroclaw, 14A F. Joliot-Curie St., 50-383 Wroclaw, Poland.
Paulina
P
Sosicka
Laboratory of Biochemistry, Faculty of Biotechnology, University of Wroclaw, 14A F. Joliot-Curie St., 50-383 Wroclaw, Poland.
Dorota
D
Maszczak-Seneczko
Laboratory of Biochemistry, Faculty of Biotechnology, University of Wroclaw, 14A F. Joliot-Curie St., 50-383 Wroclaw, Poland.
Bożena
B
Bazan
Laboratory of Biochemistry, Faculty of Biotechnology, University of Wroclaw, 14A F. Joliot-Curie St., 50-383 Wroclaw, Poland.
Yauhen
Y
Shauchuk
Laboratory of Biochemistry, Faculty of Biotechnology, University of Wroclaw, 14A F. Joliot-Curie St., 50-383 Wroclaw, Poland.
Beata
B
Kaczmarek
Laboratory of Biochemistry, Faculty of Biotechnology, University of Wroclaw, 14A F. Joliot-Curie St., 50-383 Wroclaw, Poland. Electronic address: Mariusz.Olczak@uwr.edu.pl.
Mariusz
M
Olczak
D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport / Q000235:genetics; D019556:COS Cells; D050260:Carbohydrate Metabolism / Q000235:genetics; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D004285:Dogs; D057809:HEK293 Cells; D056945:Hep G2 Cells; D006801:Humans; D061985:Madin Darby Canine Kidney Cells; D009004:Monosaccharide Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D033704:Nucleotide Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D017386:Sequence Homology, Amino Acid
D009004:Monosaccharide Transport Proteins; D033704:Nucleotide Transport Proteins; C000621190:SLC35A4 protein, human
Endoplasmic reticulum; Glycosylation; Golgi apparatus; Nucleotide sugar transporters; SLC35A subfamily