Pathogenic bacteria possess virulence proteins which are exquisitely tuned to modulate an array of targets. The type III secretion (TTS) system is broadly utilized by Gram-negative bacteria to deliver these effectors into host cells. Yersinia pseudotuberculosis delivers six virulence proteins, called "Yops," into host cells. Yops modulate the inflammatory response, cell survival, and phagocytosis–effective tactics which enable the bacterium to colonize phagocyte-rich lymph nodes. YopE is particularly well-studied, and disables members of the Rho protein family, which are critical to phagocytosis through their modulation of the actin cytoskeleton. Like many TTS effectors, translocation of YopE into host cells requires its prior association with a dedicated chaperone protein within the bacterium. The mechanism by which this association promotes YopE translocation is the topic of this dissertation. I present evidence that SycE promotes a disorder-to-order transition within the chaperone-binding region of YopE. Among the secondary structure generated upon association between SycE and YopE is a β-hairpin in YopE, and I hypothesize that the ordering of specific residues within this domain serves to target the effector to a component of the TTS system required for translocation. Structures of diverse TTS effectors from several species reveal that this motif is conserved, indicating that this motif might function as a general targeting motif. I mutated a set of three or five residues within the YopE β-hairpin and find the translocation of the altered protein to be dramatically reduced. We demonstrate that these mutations do not destabilize the YopE-SycE interaction, and we propose that the ordering of these YopE residues by the chaperone SycE identifies the effector as a substrate for the TTS system. We seek to identify the receptor for this targeting signal, and we present preliminary results on purification of several proteins that might function as receptors for translocation targeting signals. Finally, recommendations are made for future investigations.