Posttranslational Modifications of the Photoreceptor-Specific ABC Transporter ABCA4

• Published in: Biochemistry (Easton) Vol. 50; no. 32; pp. 6855 - 6866
• Main Authors: Tsybovsky, Yaroslav, Wang, Benlian, Quazi, Faraz, Molday, Robert S, Palczewski, Krzysztof
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 16.08.2011
• Subjects: Adenosine Triphosphatases - metabolism; Amino Acid Sequence; Animals; ATP-Binding Cassette Transporters - chemistry; ATP-Binding Cassette Transporters - genetics; ATP-Binding Cassette Transporters - isolation & purification; ATP-Binding Cassette Transporters - metabolism; Carbohydrate Sequence; Cattle; Electrophoresis, Polyacrylamide Gel; Glycosylation; Humans; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Phosphorylation; Protein Processing, Post-Translational; Rod Cell Outer Segment - metabolism; Tandem Mass Spectrometry
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi200774w

ABCA4 is a photoreceptor-specific ATP-binding cassette transporter implicated in the clearance of all-trans-retinal produced in the retina during light perception. Multiple mutations in this protein have been linked to Stargardt disease and other visual disorders. Here we report the first systematic study of posttranslational modifications in native ABCA4 purified from bovine rod outer segments. Seven N-glycosylation sites were detected in exocytoplasmic domains 1 and 2 by mass spectrometry, confirming the topological model of ABCA4 proposed previously. The modifying oligosaccharides were relatively short and homogeneous, predominantly representing a high-mannose type of N-glycosylation. Five phosphorylation sites were detected in cytoplasmic domain 1, with four of them located in the linker “regulatory-like” region conserved among ABCA subfamily members. Contrary to published results, phosphorylation of ABCA4 was found to be independent of light. Using human ABCA4 mutants heterologously expressed in mammalian cells, we showed that the Stargardt disease-associated alanine mutation in the phosphorylation site at position 901 led to protein misfolding and degradation. Furthermore, replacing the S1317 phosphorylation site reduced the basal ATPase activity of ABCA4, whereas an alanine mutation in either the S1185 or T1313 phosphorylation site resulted in a significant decrease in the all-trans-retinal-stimulated ATPase activity without affecting the basal activity, protein expression, or localization. In agreement with this observation, partial dephosphorylation of native bovine ABCA4 led to reduction of both basal and stimulated ATPase activity. Thus, we present the first evidence that phosphorylation of ABCA4 can regulate its function.