Zinc is an essential cofactor for many proteins. A key mechanism of zinc homeostasis during deficiency is “zinc sparing” in which specific zinc-binding proteins are repressed to reduce the cellular requirement. In this report, we evaluated zinc sparing across the zinc proteome of Saccharomyces cerevisiae. The yeast zinc proteome of 582 known or potential zinc-binding proteins was identified using a bioinformatics analysis that combined global domain searches with local motif searches. Protein abundance was determined by mass spectrometry. In zinc-replete cells, we detected over 2500 proteins among which 229 were zinc proteins. Based on copy number estimates and binding stoichiometries, a replete cell contains ~9 million zinc-binding sites on proteins. During zinc deficiency, many zinc proteins decreased in abundance and the zinc-binding requirement decreased to ~5 million zinc atoms per cell. Many of these effects were due at least in part to changes in mRNA levels rather than simply protein degradation. Measurements of cellular zinc content showed that the level of zinc atoms per cell dropped from over 20 million in replete cells to only 1.7 million in deficient cells. These results confirmed the ability of replete cells to store excess zinc and suggested that the majority of zinc-binding sites on proteins in deficient cells are either unmetalated or mismetalated. Our analysis of two abundant zinc proteins, Fba1 aldolase and Met6 methionine synthetase, supported that hypothesis. Thus, we have discovered widespread zinc sparing mechanisms and obtained evidence of a high accumulation of zinc proteins that lack their cofactor during deficiency.

Successful replication of influenza virus requires the coordinated expression of viral genes and replication of the genome by the viral polymerase, composed of the subunits PA, PB1, and PB2. Polymerase activity is regulated by both viral and host factors, yet the mechanisms of regulation and how they contribute to viral pathogenicity and tropism are poorly understood. To characterize these processes, we created a series of mutants in the 627 domain of the PB2 subunit. This domain contains a conserved "P[F/ P]AAAPP" sequence motif and the well-described amino acid 627, whose identity regulates host range. A lysine present at position 627 in most mammalian viral isolates creates a basic face on the domain surface and confers high-level activity in humans compared to the glutamic acid found at this position in avian isolates. Mutation of the basic face or the P[F/P]AAAPP motif impaired polymerase activity, assembly of replication complexes, and viral replication. Most of these residues are required for general polymerase activity, whereas PB2 K586 and R589 were preferentially required for function in human versus avian cells. Thus, these data identify residues in the 627 domain and other viral proteins that regulate polymerase activity, highlighting the importance of the surface charge and structure of this domain for virus replication and host adaptation. IMPORTANCE Influenza virus faces barriers to transmission across species as it emerges from its natural reservoir in birds to infect mammals.The viral polymerase is an important regulator of this process and undergoes discrete changes to adapt to replication in mammals. Many of these changes occur in the polymerase subunit PB2. Here we describe the systematic analysis of a key region in PB2 that controls species-specific polymerase activity. We report the importance of conserved residues that contribute to the overall charge of the protein as well as those that likely affect protein structure. These findings provide further insight into the molecular events dictating species-specific polymerase function and viral replication.

1The Zap1 transcription factor of Saccharomyces cerevisiae is a key regulator in the genomic 2 responses to zinc deficiency. Among the genes regulated by Zap1 during zinc deficiency is the 3 autophagy-related gene ATG41. Here, we report that Atg41 is required for growth in zinc-4 deficient conditions but not when zinc is abundant or when other metals are limiting. 5Consistent with a role for Atg41 in macroautophagy, we show that nutritional zinc deficiency 6 induces autophagy and that mutation of ATG41 diminishes that response. Several experiments 7 indicated that the importance of ATG41 function to growth during zinc deficiency is not 8 because of its role in macroautophagy but rather is due to one or more autophagy-independent 9 functions. For example, rapamycin treatment fully induced autophagy in zinc-deficient atg41Δ 10 mutants but failed to improve growth. In addition, atg41Δ mutants showed a far more severe 11 growth defect than any of several other autophagy mutants tested, and atg41Δ mutants 12 showed increased Hsf1 activity, an indicator of protein homeostasis stress, while other 13 autophagy mutants did not. An autophagy-independent function for ATG41 in sulfur 14 metabolism during zinc deficiency was suggested by analyzing the transcriptome of atg41Δ 15 mutants during the transition from zinc-replete to deficient conditions. Analysis of sulfur 16 metabolites confirmed that Atg41 is needed for the normal accumulation of methionine, 17 homocysteine, and cysteine in zinc-deficient cells. Therefore, we conclude that Atg41 plays 18 roles in both macroautophagy and sulfur metabolism during zinc deficiency. 19 20 4