Involvement of the Glucose-Regulated Protein 94 (Dd-GRP94) in Starvation Response of Dictyostelium discoideum Cells Tsuyoshi Morita, 1 Kenji Saitoh, Takashi Takagi, and Yasuo Maeda Biological Institute, Graduate School of Science, Tohoku University, Aoba, Sendai 980-8578, Japan Received June 15, 2000 Upon deprivation of nutrients, Dictyostelium discoi- deum Ax-2 cells arrest proliferation and initiate a metamorphosed developmental program including in- duction of altered gene expressions which are neces- sary for differentiation. In Ax-2 cells, we found out a member of Hsp90 family usually contained in the en- doplasmic reticulum (ER), Dd-GRP94 (Dictyostelium discoideum glucose-regulated protein 94). In general, GRP94 are induced either by glucose-depletion or by depletion of Ca 2 in intracellular Ca 2 stores. Unex- pectedly, however, the expression of Dd-grp94 was greatly reduced within 60 min of starvation. Dd-grp94- overexpressing cells (GRP94 OE cells) collected without forming distinct aggregation streams, and never formed normal fruiting bodies. Also, prespore differ- entiation as well as maturation into spores and stalk cells were particularly impaired in the GRP94 OE cells. Thus Dd-GRP94 seems to be crucial in late differ- entiation as well as in starvation response. © 2000 Academic Press Key Words: growth; starvation; differentiation; GRP94; endoplasmic reticulum; Dictyostelium discoideum. The development of Dictyostelium discoideum pro- vides us an excellent system for investigating the re- lationship between growth and differentiation. Vegeta- tive cells grow and proliferate as free-living amoeboid cells as long as external nutrients are available. Upon deprivation of nutrients, however, starving cells progress through the cell cycle to a particular check- point (referred to as the putative shift point; PS-point) in the mid-late G2 phase of cell cycle and enter the differentiation phase from this point (1). As initial events of differentiation from the PS-point, cells ac- quire aggregation competence and aggregate by che- motaxis to cAMP (2) and EDTA-resistant cohesiveness (3). The aggregate forms a migrating slug that eventu- ally develops to a fruiting body consisting of a mass of spores and a supporting cellular stalk. Within the slug, a clear differentiation pattern with the anterior prestalk/posterior prespore cells has been recognized. Thus growth and differentiation are temporally sepa- rated from each other and easily controlled by nutri- tional conditions. It has been demonstrated that the phosphorylation levels of 90 kDa and 101 kDa phosphoproteins are specifically reduced in response to differentiation from the PS-point, and that the 32 kDa phosphoprotein is completely dephosphotylated under conditions of nu- tritional deprivation (4). K252a, a potent inhibitor of protein kinases, was found to promote the progress of development after starvation when applied to cells lo- cated around the PS-point, possibly by inducing the phase-shift of 48 kDa to 50 kDa phosphoprotein (5). Recently, we have found that the 90 kDa phosphopro- tein virtually consists of two components (90 kDa and 92 kDa phosphoproteins). Interestingly, the 90 kDa phosphoprotein has been shown to be the heat shock cognate protein 90 (Hsc90), cytosolic Hsp90 in Dictyo- stelium discoideum (Saito et al., personal communica- tion). As presented here, the 92 kDa phosphoprotein has an amino acid sequence “EFRSILYIPEN” well con- served in the members of Hsp90 family but is different from the D. discoideum Hsc90. In general, Hsp90 is a well-conserved major molecular chaperone and ubiqui- tous in the cytosol and the endoplasmic reticulum (ER), relating to the cell cycle regulation and signal trans- duction in cell differentiation or apoptosis. From ho- mology search of the 92 kDa phosphoprotein, it was concluded to be Dd-GRP94 (Dictyostelium discoideum glucose-regulated protein 94), the endoplasmic reticu- lum Hsp90. In general, the expression of grp94 gene is induced by a variety of stress conditions, such as glucose-depletion (6, 7) and Ca 2+ depletion in the ER (8). During the ER stress, GRP94 associates with other numerous molecular chaperones (grp78 (Bip), calreti- 1 To whom correspondence should be addressed. Fax: 080-22-217- 6709. E-mail: tsuyo@biology.tohoku.ac.jp. Biochemical and Biophysical Research Communications 274, 323–331 (2000) doi:10.1006/bbrc.2000.3096, available online at http://www.idealibrary.com on 323 0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.