Structure-function analysis of the BEACH protein LvsA.
Most eukaryotes have several members of the BEACH family of proteins but the molecular function of these large proteins remains unknown. The Dictyostelium BEACH protein LvsA is essential for cytokinesis and contractile vacuole activity. The functional contribution of different portions of LvsA was tested here by deletion analysis. The C-terminal WD domain was important for protein stability and C-terminal deletions resulted in loss of LvsA function. In contrast, N-terminal deletions yielded abundant protein expression that could be assayed for function. Despite very low sequence conservation of the N-terminal portion of LvsA, this region is important for its function in vivo. Deletion of 689 N-terminal amino acids produced a protein that was functional in cytokinesis but partially functional in osmoregulation. Further deletions resulted in the complete loss of LvsA function. Using in vitro fractionation assays we found that LvsA sedimented with membranes but that this association does not require the N-terminal portion of LvsA. Interestingly, the association of LvsA with the contractile vacuole was perturbed by the loss of drainin, a protein important for vacuole function. In drainin-null cells, LvsA bound irreversibly to engorged contractile vacuoles that fail to expel water. These experiments help delineate the biochemical and physiological requirements for function of one important BEACH protein, LvsA.
Wu
Wei-I
WI
Section of Molecular Cell & Developmental Biology and Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712, USA.
Yajnik
Juhi
J
Siano
Michael
M
De Lozanne
Arturo
A
D000818:Animals; D001483:Base Sequence; D002458:Cell Fractionation; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D048749:Cytokinesis; D004023:Dictyostelium / Q000737:chemistry / Q000166:cytology / Q000235:genetics; D008565:Membrane Proteins / Q000378:metabolism; D011485:Protein Binding; D015800:Protozoan Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion / Q000235:genetics; D013329:Structure-Activity Relationship; D014617:Vacuoles / Q000378:metabolism; D014882:Water-Electrolyte Balance
C403705:LvsA protein, Dictyostelium; D008565:Membrane Proteins; D015800:Protozoan Proteins; C120615:drainin