Abstract
BackgroundSEDLIN, a 140 amino acid subunit of the Transport Protein Particle (TRAPP) complex, is ubiquitously expressed and interacts with the transcription factors c-myc promoter-binding protein 1 (MBP1), pituitary homeobox 1 (PITX1) and steroidogenic factor 1 (SF1). SEDLIN mutations cause X-linked spondyloepiphyseal dysplasia tarda (SEDT).Methodology/Principal FindingsWe investigated the effects of 4 missense (Asp47Tyr, Ser73Leu, Phe83Ser and Val130Asp) and the most C-terminal nonsense (Gln131Stop) SEDT-associated mutations on interactions with MBP1, PITX1 and SF1 by expression in COS7 cells. Wild-type SEDLIN was present in the cytoplasm and nucleus and interacted with MBP1, PITX1 and SF1; the SEDLIN mutations did not alter these subcellular localizations or the interactions. However, SEDLIN was found to homodimerize, and the formation of dimers between wild-type and mutant SEDLIN would mask a loss in these interactions. A mammalian SEDLIN null cell-line is not available, and the interactions between SEDLIN and the transcription factors were therefore investigated in yeast, which does not endogenously express SEDLIN. This revealed that all the SEDT mutations, except Asp47Tyr, lead to a loss of interaction with MBP1, PITX1 and SF1. Three-dimensional modelling studies of SEDLIN revealed that Asp47 resides on the surface whereas all the other mutant residues lie within the hydrophobic core of the protein, and hence are likely to affect the correct folding of SEDLIN and thereby disrupt protein-protein interactions.Conclusions/SignificanceOur studies demonstrate that SEDLIN is present in the nucleus, forms homodimers and that SEDT-associated mutations cause a loss of interaction with the transcription factors MBP1, PITX1 and SF1.
Highlights
SEDLIN, which is one of the subunits of the Transport Protein Particle (TRAPP) complex, is involved in the targeting and fusion of endoplasmic reticulum (ER)-derived transport vesicles to the Golgi acceptor compartment [1]
myc promoter-binding protein 1 (MBP1), pituitary homeobox 1 (PITX1) and steroidogenic factor 1 (SF1) were expressed in the nucleus and cytoplasm, but PITX1 and SF1 co-localized with SEDLIN in the nucleus only, whereas MBP1 co-localized with SEDLIN in the nucleus and cytoplasm (Figure 3B)
Our results show that SEDLIN forms homodimers (Figures 6 and 7), is localized to the cytoplasm and nucleus (Figure 3), and that wild-type SEDLIN co-localizes and interacts with the transcription factors MBP1, PITX1 and SF1 (Figures 3, 4 and 7)
Summary
SEDLIN, which is one of the subunits of the Transport Protein Particle (TRAPP) complex, is involved in the targeting and fusion of endoplasmic reticulum (ER)-derived transport vesicles to the Golgi acceptor compartment [1]. SEDLIN has been reported to interact with proteins that are not part of the TRAPP complex, but are either transcription factors, such as the c-myc promoterbinding protein 1 (MBP1), pituitary homeobox 1 (PITX1) and steroidogenic factor 1 (SF1) [3,4], or the intracellular chloride channels, CLIC1 and CLIC2 [5]. We focused on the 4 SEDT-associated missense mutations (Figure 1) as these were predicted to yield a full-length protein and were not likely to affect the tertiary structure of SEDLIN substantially, as well as the most C-terminal nonsense (Gln131Stop) mutation, which if translated, is predicted to result in the loss of the last ten amino acids. SEDLIN, a 140 amino acid subunit of the Transport Protein Particle (TRAPP) complex, is ubiquitously expressed and interacts with the transcription factors c-myc promoter-binding protein 1 (MBP1), pituitary homeobox 1 (PITX1) and steroidogenic factor 1 (SF1).
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