Wiskott-Aldrich Syndrome protein (WASP) is defective in Wiskott-Aldrich Syndrome, an inherited immunodeficiency disorder. WASP promotes assembly of branched actin filaments and functions in a complex with WASP-interacting protein (WIP). Both proteins are proline-rich and bind the Src-homology 3 (SH3) domains of several other cytoskeletal proteins to facilitate signaling to the actin cytoskeleton. The yeast Saccharomyces cerevisiae has an actin cytoskeleton similar to mammalian cells and the homologues of WASP and WIP in S. cerevisiae are Las17p and Vrp1p. In yeast, Vrp1p binds the SH3 domain of Hof1p, a key regulator of the yeast cytoskeleton, and this binding promotes cytokinesis while a lack of Vrp1p inhibits cytokinesis. This study investigates the possibility that the interaction between Vrp1p and the Hof1p SH3 domain has been conserved in humans. This study proposes that human WIP, which is known to function in yeast cells lacking Vrp1p, binds the yeast Hof1p SH3 domain through proline-rich motifs and thereby counteracts an inhibitory effect of the unbound yeast Hof1p SH3 domain on cytokinesis. It was found that the yeast Hof1p SH3 domain interacts with two proline-rich motifs (PRMs) in the C-terminus of human WIP. Yeast two-hybrid interaction tests and in vitro pull-down assays both confirmed the motif PRM1 (PATPQLPSRS) as one of the interaction sites. More yeast two-hybrid interaction tests revealed the second PRM (PRM2) mediating interaction to be PPPPPSTS, a PRM that has been previously reported to be required for the rescue of cytoskeletal defects in yeast cells lacking Vrp1p. However, this result could not be confirmed in in vitro pull-down assays. The other PRM essential for interaction between the C-terminus of human WIP and the Hof1p SH3 domain was found to be PRM3 (PPLPPIPR), which is located at the extreme C-terminus of human WIP. Unexpectedly, yeast cells lacking Vrp1p but expressing mutated full-length human WIP lacking PRM1 and PRM2 or PRM1 and PRM3 did not exhibit any cytoskeletal defects at elevated temperatures (a stress condition). A yeast twohybrid interaction test with these mutated full-length human WIP constructs revealed that, despite the deletion of the PRMs in the C-terminus of human WIP mediating Hof1p SH3 domain interaction, the full-length WIP protein was still able to interact with the Hof1p SH3 domain. This study provides evidence that the interaction between Vrp1p and the Hof1p SH3 domain in yeast has been conserved in humans. Two PRMs were found in the C-terminus of human WIP that mediate interaction with the yeast Hof1p SH3 domain. Further, the human homologue of Hof1p, PSTPIP1, interacted with the same C-terminal fragment of human WIP in the yeast two-hybrid interaction test demonstrating that a similar mechanism exists in humans. In the absence of WIP, the unbound SH3 domain of Hof1p (or PSTPIP1 in humans) might be the cause cytoskeletal defects possibly contributing to Wiskott-Aldrich Syndrome. The results of this study help in revealing the mechanisms of cytoskeletal rearrangement in the yeast model allowing us to unravel similar mechanisms in humans in the future.