Use of expression constructs to dissect the functional domains of the CHS/beige protein: identification of multiple phenotypes.
The Chediak-Higashi Syndrome (CHS) and the orthologous murine disorder beige are characterized at the cellular level by the presence of giant lysosomes. The CHS1/Beige protein is a 3787 amino acid protein of unknown function. To determine functional domains of the CHS1/Beige protein, we generated truncated constructs of the gene/protein. These truncated proteins were transiently expressed in Cos-7 or HeLa cells and their effect on membrane trafficking was examined. Beige is apparently a cytosolic protein, as are most transiently expressed truncated Beige constructs. Expression of the Beige construct FM (amino acids 1-2037) in wild-type cells led to enlarged lysosomes. Similarly, expression of a 5.5-kb region (amino acids 2035-3787) of the carboxyl terminal of Beige (22B) also resulted in enlarged lysosomes. Expression of FM solely affected lysosome size, whereas expression of 22B led to alterations in lysosome size, changes in the Golgi and eventually cell death. The two constructs could be used to further dissect phenotypes resulting from loss of the Beige protein. CHS or beigej fibroblasts show an absence of nuclear staining using a monoclonal antibody directed against phosphatidylinositol 4,5 bisphosphate [PtdIns(4,5) P2]. Transformation of beige j fibroblasts with a YAC containing the full-length Beige gene resulted in the normalization of lysosome size and nuclear PtdIns(4,5)P2 staining. Expression of the carboxyl dominant negative construct 22B led to loss of nuclear PtdIns(4,5)P2 staining. Expression of the FM dominant negative clone did not alter nuclear PtdIns(4,5) P2 localization. These results suggest that the Beige protein interacts with at least two different partners and that the Beige protein affects cellular events, such as nuclear PtdIns(4,5)P2 localization, in addition to lysosome size.
Ward
Diane McVey
DM
Department of Pathology, School of Medicine, University of Utah, Salt Lake City, Utah 84132, USA.
Shiflett
Shelly L
SL
Huynh
Dinh
D
Vaughn
Michael B
MB
Prestwich
Glenn
G
Kaplan
Jerry
J
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D019556:COS Cells; D002609:Chediak-Higashi Syndrome; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D017931:DNA Primers; D005347:Fibroblasts / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008247:Lysosomes / Q000648:ultrastructure; D051379:Mice; D008969:Molecular Sequence Data; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D033921:Vesicular Transport Proteins
D017931:DNA Primers; D047908:Intracellular Signaling Peptides and Proteins; C102161:LYST protein, human; C491133:Lyst protein, mouse; D019269:Phosphatidylinositol 4,5-Diphosphate; D011506:Proteins; D011994:Recombinant Proteins; D033921:Vesicular Transport Proteins