In the present study, we employed genetic and biochemical approaches and demonstrated that in the gcp operon of S. aureus, the essential Gcp protein interacts with another essential protein YeaZ. More importantly, we identified key domains of Gcp that are not only required for Gcp binding to YeaZ, but also play important roles in Gcp's essentiality for bacterial growth. Our results suggest that the interaction of Gcp and YeaZ may contribute to the essentiality of Gcp. In addition, our results showed that there was neither O-sialoglycoprotein endopeptidase activity nor Gcp specific protease activity detected for the purified recombinant staphylococcal Gcp and/or YeaZ, suggesting that the staphylococcal Gcp and YeaZ may function differently compared to their homologs in M. hemolytica and E. coli, respectively. These findings may provide new insights into the molecular mechanisms and biological function of the essential protein, Gcp.
The identification of the staphylococcal Gcp-YeaZ interaction is consistent with the previous report that the Gcp homolog in E. coli
, YgjD, binds to YeaZ the homolog of SA1856 
. However, in E. coli
another essential protein, YjeE, also interacts with YeaZ, suggesting that YjeE may function as a regulator to modulate YeaZ-YgjD interaction, and that the YjeE-YeaZ-YgjD network may be involved in an essential cellular process 
. In contrast, in S. aureus
there is no report indicating that the homolog of YjeE, SA1857, is indispensable for bacterial viability 
. We are in the process of examining the requirement of SA1857 and SA1855 for the growth of S. aureus
. Furthermore, in our Y2H studies we found no evidence that SA1857 and SA1855 interacts with either Gcp or YeaZ. Although the staphylococcal YeaZ and Gcp proteins have approximately 26% and 42% identity compared to E. coli
YeaZ and YgjD sequences, respectively, our studies indicate that Gcp and YeaZ likely function differently or interact with different partner proteins compared to their homologs in E. coli
. We observed that in Y2H assays the growth of yeast cells on SC-His plates seems to be faster than on SC-Ade plates. This is likely due to the stringent growth difference between SC-Ade and SC-His selective medium 
. Although we unveiled that the mutation of Y317-F322 region eliminated the ability of Gcp to bind YeaZ, and the mutations of D324-N329 and S332-Y336 regions alleviated the capacity of Gcp to bind YeaZ, the attenuated binding ability may be attributable to the altered tertiary conformation of Gcp. To determine the potential impact of topology for mutated Gcp proteins is beyond scope of the present study. However, we are currently working to further characterize and define the interaction of Gcp and YeaZ and identify the critical residue(s) for Gcp to bind YeaZ and vice versa.
In both yeast and archaea, Kae1p (Gcp homolog) directly interacts with a Bud32p kinase, inhibiting the kinase activity, which is required both for transcription and the telomere homeostatic function of the endopeptidase-like kinase chromatin-associated (EKC)/kinase, endopeptidase, and other peptidases of small size (KEOPS) in yeast cells 
. In eukaryotes, the Gcp homolog is Qri7 in yeast and OSGEPL1 (O
-sialoglycoprotein endopeptidase) in worm and human. Qri7 and OSGEPL1 anchor to the mitochondrial inner membrane and are essential for the maintenance of mitochondrial genome 
. Qri7 is able to complement the depletion of E. coli
YgjD (homolog of Gcp), whereas Kae1 fails to do so, suggesting the functional similarity between Qri7 and YgjD 
. Although bacteria lack a Bud32 homolog, YeaZ may substitute for Bud32 to form a functional complex with Gcp. Our complementation experiments established that the mutations of Gcp that disrupt the Gcp-YeaZ interaction could not complement the depletion of endogenous Gcp. We also observed that the deletion of the C-terminal residues, E337–E341, slightly impaired the ability of Gcp to interact with YeaZ (); and consequently, this truncated Gcp (Gcpseg1) delayed its complementary effect on bacterial growth (longer lag-phase of growth). These data suggest that the staphylococcal Gcp-YeaZ interaction may play an important role in Gcp's essentiality; thus the interruption of Gcp-YeaZ interaction may be utilized as a novel mode of action for developing new classes antibacterial agents, partially against MRSA or VRSA caused infections. It is known that bacterial autolysis is important for bacterial cell division and growth. Previously, we have demonstrated that the staphylococcal Gcp is a critical modulator of bacterial autolysis 
; however, the mechanism of regulation of autolysis by Gcp remains to be determined; it is necessary to explore the potential role of the Gcp-YeaZ interaction in the process of autolysis.
Our purified recombinant staphylococcal Gcp and YeaZ did not appear to have an O
-sialoglycoprotein endopeptidase activity against glycophorin A. We also employed an alternative approach to examine the potential glycoprotease activity by measuring cell associated ligand, PSGL1 (P-selectin glycoprotein ligand 1), using FACS. Neither the purified recombinant staphylococcal Gcp, YeaZ, nor the concentrated supernatants of S. aureus
culture appeared to have the glycoprotease activity (data not shown). Our results are in agreement with previous reports that YgjD (homolog of Gcp in E. coli
) and Kae1 (homolog of Gcp in Pyrococcus abyssi
) do not exhibit any endopeptidase activity 
. In addition, E. coli YeaZ is able to specifically cleave YgjD (the homolog of Gcp), whereas consistent with S. typhimurium
our purified soluble recombinant staphylococcal YeaZ did not exhibit such a protease activity. The lack of protease activity of purified recombinant Gcp and YeaZ may result from potential modifications during expression and purification process. However, it is likely that the Gcp and YeaZ homologs may have different biological functions among different species, because sequence alignment analysis revealed that the staphylococcal Gcp does not possess any E. coli
YeaZ cleavage sites, including K171- L172 and F195-V196 residues that were identified in YgjD 
. In addition, we cannot exclude the possibility that the staphylococcal Gcp and YeaZ proteins may possess a specific proteolytic activity against a substrate in a process that is critical for the viability of S. aureus
In conclusion, we demonstrated within the four novel proteins encoded by the gcp operon, the essential Gcp interacts with another essential protein YeaZ of S. aureus. Moreover, we identified the C-terminal Y317-F322, D324-N329, and S332-Y336 regions to be important for Gcp to bind YeaZ, as well as for Gcp's essentiality, whereas the C-terminal E337–E344 region is dispensable for the staphylococcal Gcp-YeaZ interaction and Gcp's essentiality. These data suggest that the interaction of Gcp and YeaZ may at least partially contribute to the essentiality of Gcp for S. aureus growth. Our findings provide new insights into the potential mechanisms and biological function of the novel essential protein, Gcp, as well as potential novel targets for the development of new classes antibacterial agents.