Characterization of SCP-2 from Euphorbia lagascae reveals that a single Leu/Met exchange enhances sterol transfer activity.
Sterol carrier protein-2 (SCP-2) is a small intracellular basic protein domain implicated in peroxisomal beta-oxidation. We extend our knowledge of plant SCP-2 by characterizing SCP-2 from Euphorbia lagascae. This protein consists of 122 amino acids including a PTS1 peroxisomal targeting signal. It has a molecular mass of 13.6 kDa and a pI of 9.5. It shares 67% identity and 84% similarity with SCP-2 from Arabidopsis thaliana. Proteomic analysis revealed that E. lagascae SCP-2 accumulates in the endosperm during seed germination. It showed in vitro transfer activity of BODIPY-phosphatidylcholine (BODIPY-PC). The transfer of BODIPY-PC was almost completely inhibited after addition of phosphatidylinositol, palmitic acid, stearoyl-CoA and vernolic acid, whereas sterols only had a very marginal inhibitory effect. We used protein modelling and site-directed mutagenesis to investigate why the BODIPY-PC transfer mediated by E. lagascae SCP-2 is not sensitive to sterols, whereas the transfer mediated by A. thaliana SCP-2 shows sterol sensitivity. Protein modelling suggested that the ligand-binding cavity of A. thaliana SCP-2 has four methionines (Met12, 14, 15 and 100), which are replaced by leucines (Leu11, 13, 14 and 99) in E. lagascae SCP-2. Changing Leu99 to Met99 was sufficient to convert E. lagascae SCP-2 into a sterol-sensitive BODIPY-PC-transfer protein, and correspondingly, changing Met100 to Leu100 abolished the sterol sensitivity of A. thaliana SCP-2.
Viitanen
Lenita
L
Department of Biochemistry and Pharmacy, Abo Akademi University, Turku, Finland.
Nylund
Matts
M
Eklund
D Magnus
DM
Alm
Christina
C
Eriksson
Ann-Katrin
AK
Tuuf
Jessica
J
Salminen
Tiina A
TA
Mattjus
Peter
P
Edqvist
Johan
J
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D017360:Arabidopsis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001692:Biological Transport / Q000235:genetics; D001896:Boron Compounds / Q000737:chemistry; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D028481:Euphorbia / Q000235:genetics*; D018506:Gene Expression Regulation, Plant; D018525:Germination; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D019308:Palmitic Acid / Q000737:chemistry / Q000378:metabolism; D010713:Phosphatidylcholines / Q000737:chemistry / Q000378:metabolism / Q000493:pharmacokinetics; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011487:Protein Conformation; D012639:Seeds / Q000201:enzymology; D016415:Sequence Alignment; D013261:Sterols / Q000737:chemistry / Q000378:metabolism*
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D002352:Carrier Proteins; D010713:Phosphatidylcholines; D010940:Plant Proteins; D013261:Sterols; C072087:sterol carrier proteins; D019308:Palmitic Acid