First published online April 27, 2007; 10.1105/tpc.106.049346
The Plant Cell 19:1295-1312 (2007)
© 2007 American Society of Plant Biologists
The Arabidopsis AAA ATPase SKD1 Is Involved in Multivesicular Endosome Function and Interacts with Its Positive Regulator LYST-INTERACTING PROTEIN5[W]
Thomas J. Haasa,1,
Marek K. Sliwinskia,1,
Dana E. Martínezb,
Mary Preussc,
Kazuo Ebined,
Takashi Uedad,
Erik Nielsenc,2,
Greg Odorizzie and
Marisa S. Oteguia,3
a Department of Botany, University of Wisconsin, Madison, Wisconsin 53706
b Instituto de Fisiología Vegetal, Universidad Nacional de La Plata, 1900 La Plata, Argentina
c Donald Danforth Plant Science Center, St. Louis, Missouri 63132
d Department of Biological Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan
e Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado 80309-0347
3 To whom correspondence should be addressed. E-mail otegui{at}wisc.edu; fax 608-262-7509.
In yeast and mammals, the AAA ATPase Vps4p/SKD1 (for Vacuolar protein sorting 4/SUPPRESSOR OF K+ TRANSPORT GROWTH DEFECT1) is required for the endosomal sorting of secretory and endocytic cargo. We identified a VPS4/SKD1 homolog in Arabidopsis thaliana, which localizes to the cytoplasm and to multivesicular endosomes. In addition, green fluorescent protein–SKD1 colocalizes on multivesicular bodies with fluorescent fusion protein endosomal Rab GTPases, such as ARA6/RabF1, RHA1/RabF2a, and ARA7/RabF2b, and with the endocytic marker FM4-64. The expression of SKD1E232Q, an ATPase-deficient version of SKD1, induces alterations in the endosomal system of tobacco (Nicotiana tabacum) Bright Yellow 2 cells and ultimately leads to cell death. The inducible expression of SKD1E232Q in Arabidopsis resulted in enlarged endosomes with a reduced number of internal vesicles. In a yeast two-hybrid screen using Arabidopsis SKD1 as bait, we isolated a putative homolog of mammalian LYST-INTERACTING PROTEIN5 (LIP5)/SKD1 BINDING PROTEIN1 and yeast Vta1p (for Vps twenty associated 1 protein). Arabidopsis LIP5 acts as a positive regulator of SKD1 by increasing fourfold to fivefold its in vitro ATPase activity. We isolated a knockout homozygous Arabidopsis mutant line with a T-DNA insertion in LIP5. lip5 plants are viable and show no phenotypic alterations under normal growth conditions, suggesting that basal SKD1 ATPase activity is sufficient for plant development and growth.
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