A family of proteins that stabilize the Ran/TC4 GTPase in its GTP-bound conformation

Abstract

Ran/TC4, referred to here as Ran1, is a 25-kilodalton nuclear GTP-binding protein with an acidic C terminus that lacks any consensus prenylation sites. Here, we use a nitrocellulose overlay assay to identify potential effector proteins that bind specifically and with high affinity to the GTP-bound form of Ran1. GTP-Ran1 is shown to bind a variety of proteins, present in many eukaryotic tissues and cell extracts. A 28-kDa protein is cytosolic, whereas others, consisting of proteins of 86-300 kDa, are primarily localized in the nucleus. Binding is highly specific and is not detected by other small GTPases, such as c-Ha-Ras or Rab3A. Both deletion of the C-terminal-DEDDDL acidic sequence or alteration of the N terminus of Ran1 inhibits binding. However, these altered forms of Ran1 maintain the capacity to bind guanyl nucleotides and interact with the nucleotide exchange factor. The Ran1-binding proteins potently inhibit release of GTP from Ran1. These proteins can therefore maintain Ran1 in the "on" state and are potential down-stream effectors for Ran1-dependent cellular processes.