S. Jones et al., REQUIREMENT OF NUCLEOTIDE EXCHANGE FACTOR FOR YPT1 GTPASE MEDIATED PROTEIN-TRANSPORT, The Journal of cell biology, 130(5), 1995, pp. 1051-1061
Small GTPases of the rab family are involved in the regulation of vesi
cular transport. It is believed that cycling between the GTP- and GDP-
bound forms, and accessory factors regulating this cycling are crucial
for rab function. However, an essential role for rab nucleotide excha
nge factors has not yet been demonstrated, In this report we show the
requirement of nucleotide exchange factor activity for Ypt1 GTPase med
iated protein transport. The Ypt1 protein, a member of the rab family,
plays a role in targeting vesicles to the acceptor compartment and is
essential for the first two steps of the yeast secretory pathway. We
use two YPT1 dominant mutations that contain alterations in a highly c
onserved GTP-binding domain, N121I and D124N. YPT1-D124N is a novel mu
tation that encodes a protein with nucleotide specificity modified fro
m guanine to xanthine, This provides a tool for the study of an indivi
dual rab GTPase in crude extracts: a xanthosine triphosphate (XTP)-dep
endent conditional dominant mutation. Both mutations confer growth inh
ibition and a block in protein secretion when expressed in vivo. The p
urified mutant proteins do not bind either GDP or GTP. Moreover, they
completely inhibit the ability of the exchange factor to stimulate nuc
leotide exchange for wild type Ypt1 protein, and are potent inhibitors
of ER to Golgi transport in vitro at the vesicle targeting step, The
inhibitory effects of the Ypt1-D124N mutant protein on both nucleotide
exchange activity and protein transport in vitro can be relieved by X
TP, indicating that it is the nucleotide-free form of the mutant prote
in that is inhibitory. These results suggest that the dominant mutant
proteins inhibit protein transport by sequestering the exchange factor
from the wild type Ypt1 protein, and that this factor has an essentia
l role in vesicular transport.