SUMMARY Zinc binding metalloproteins and metalloenzymes constitute approximately 10% of the vertebrate proteome and are essential for many cellular processes. Due to the critical importance of zinc to vertebrate physiology, zinc deficiency leads to growth defects, aberrant immune function, neurological disorders, cancers, and increased risk of infection. This is a pervasive public health problem considering that approximately half of the world's population suffers from dietary zinc deficiency, now the fifth most important risk factor for mortality in developing countries. Although the clinical link between Zn deficiency and infection is established, the molecular mechanisms are not well understood. Despite the known importance of zinc cofactors to cellular function, the mechanism by which zinc is inserted into metalloproteins is poorly understood. In this application we report our discovery of zinc-regulated GTPase metalloprotein activator 1 (Zng1) as the first reported zinc metallochaperone that inserts zinc into cognate client metalloproteins. We identify the protein processing enzyme methionine aminopeptidase 1 (Metap1) and the transcription factor zinc finger homeobox 3 (Zfhx3) as Zng1 clients, and we describe a conserved domain that mediates an interaction between Zng1 and these metalloproteins. This finding forms the basis of proposed structural, biochemical, and physiological experiments to define the mechanism of Zng1-dependent metal delivery to client proteins. Single nucleotide polymorphisms in Zng1 are associated with increased incidence of severe infections in humans, establishing an important link between Zng1 and infection in the zinc starved host. Zng1-dependent metal transfer increases the functional activities of Metap1 and Zfhx3, and loss of Zng1 activity in mice and zebrafish negatively impacts organismal development and increases susceptibility to multiple infectious diseases. Based on these findings, we propose a model whereby the Zng1 family of enzymes are evolutionarily conserved metallochaperones that transfer zinc to Metap1 and Zfhx3 during conditions of extreme zinc deficiency. We predict that this process of co-factor delivery leads to regulated protein processing through Metap1 and coordinated gene expression changes through Zfhx3. Combined, these activities help orchestrate the cellular response to infection during zinc starvation. Experiments described in this proposal will test this model and determine the molecular mechanism by which Zng1 transfers metal to Metap1 and Zfhx3, define the role of Zng1-client interactions in proteostasis and transcription, and reveal the in vivo contribution of Zng1 metal delivery to vertebrate defense against infection. Collectively, findings from this proposal will uncover the biological and pathophysiological relevance of this newly identified family of zinc metallochaperones and make important contributions to nutrition and infection biology.