Among the most intriguing mysteries in contemporary biology is the functional mechanism of the vault complex. Renowned for its immense size and arched structure resembling that of “cathedral vaults,” it is found across tissue types, cellular compartments, and eukaryotic species, with a patchy pattern of conservation that stretches from slime molds to humans. Although work since vault’s initial discovery in 1986 has failed to define its precise function, studies of the vault’s primary component – the major vault protein (MVP) – have provided some clues. Research observing MVP knockout mice and condition-dependent changes in MVP expression has demonstrated vault’s ability to promote cell survival following exposure to stress. Structures of the giant oligomeric MVP “cage,” which comprises most of vault’s mass, have spurred hypotheses that it acts as a molecular “cargo” transport module and/or signaling scaffold. This supposition has been bolstered by reports of vault’s ability to rapidly relocalize to different cellular compartments following cell perturbation, as well as observations of heterogeneous density within its Fabergé egg-like cage. Lacking any enzymatic activity of its own, the vault cage most likely acts as a relatively inert vessel. We hypothesized that regulation by additional molecules is critical to MVP’s ability to accept transiently associated cargo, move between organelles, and otherwise mediate cell signaling. Some obvious candidates to fill these regulatory roles are vault’s understudied “minor” components: the enzyme poly (ADP-ribose) polymerase 4 (PARP4), the NOD-like protein telomerase component 1 (TEP1), and short non-coding transcripts known as vault RNAs (vtRNAs), all of which reside within the MVP cage. My research has therefore sought to explore the roles of vault’s minor components and expand our understanding of vault as a multispecies complex. Due to its established enzymatic activity, I decided to begin by focusing on PARP4, an ADP-ribosyltransferase that consumes the metabolite NAD+ to deposit post-translational modifications (PTMs) onto its target substrates. In Chapter 2, I report our novel complex structures, which resolve the interface between MVP and PARP4, as well as a surprising interaction between MVP and NAD+. Accounts of my initial efforts to study the cellular function of vault-bound PARP4 are described in Chapter 3. I summarize my thesis work and its import in Chapter 4. Additional data supporting the findings listed in Chapter 2 and preliminary work to elucidate the structure of the regions of PARP4 that do not interact with vault can be found in Appendices A and B, respectively.