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[Preprint]. 2023 May 17:rs.3.rs-2860473.
doi: 10.21203/rs.3.rs-2860473/v1.

TerC Proteins Function During Protein Secretion to Metalate Exoenzymes

Bixi He  1 , Ankita J Sachla  1 , John D Helmann  1
Affiliations

TerC Proteins Function During Protein Secretion to Metalate Exoenzymes

Bixi He et al. Res Sq. .

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Abstract

Cytosolic metalloenzymes acquire metals from buffered intracellular pools. How exported metalloenzymes are appropriately metalated is less clear. We provide evidence that TerC family proteins function in metalation of enzymes during export through the general secretion (Sec-dependent) pathway. Bacillus subtilis strains lacking MeeF(YceF) and MeeY(YkoY) have a reduced capacity for protein export and a greatly reduced level of manganese (Mn) in the secreted proteome. MeeF and MeeY copurify with proteins of the general secretory pathway, and in their absence the FtsH membrane protease is essential for viability. MeeF and MeeY are also required for efficient function of the Mn2+-dependent lipoteichoic acid synthase (LtaS), a membrane-localized enzyme with an extracytoplasmic active site. Thus, MeeF and MeeY, representative of the widely conserved TerC family of membrane transporters, function in the co-translocational metalation of Mn2+-dependent membrane and extracellular enzymes.

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Conflict of interest statement

DECLARATION OF INTEREST: The authors declare no competing interests.

Figures

Figure 1.
Figure 1.. MeeF and MeeY are required for efficient secretion of feeding proteases to access nutrients in tryptone.
(a) Colony size of WT, meeF, meeY, and FY on LB agar. (b) Colony size of WT and FY mutant on a defined glucose-minimal media (MM). MM agar plates were made with and without NaCl, tryptone, or yeast extract (Y.E.). Agar plates with well isolated colonies were imaged after 24 h at 37 °C, and sizes measured using Image J. 40 or more isolated colonies from at least three independent cultures were included in the measurements for each strain, data is presented as mean ± SD. **, P=0.0014; ****, P<0.0001, P value was calculated using Welch’s t test, two-tailed. Average changes were calculated as “change = (sample - control) / control * 100%”. WT is the control for each nutrient component. (c) Protease hydrolysis of different strains on 5% milk agar plates. Zone of clearance due to protease activity was imaged. (d) Extracellular protease activities in the supernatants were detected by gelatin zymography. Supernatants were collected from overnight cultures with the same cell number. Higher protease activities correspond to clearer bands on the gel matrix. M, marker; Δ7, mutant lacking seven extracellular proteases (ΔaprE, ΔnprE, ΔnprB, Δbpr, Δepr, Δmpr, Δvpr).
Figure 2.
Figure 2.. FY mutants have a generalized secretion defect.
(a) FY mutants (but not the single F and Y mutants) have reduced levels of secreted proteins in the spent medium (supernatant fraction) after overnight culture. (b) FY mutants also have dramatically reduced levels of Mn in the spent medium after overnight growth as monitored by ICP-MS analysis. For (a) and (b), samples were from three independent experiments. Data is presented as mean ± SD. ****, P < 0.0001, P value was calculated using Welch’s t test, two-tailed. (c) Silver staining showing reduced extracellular protein in the supernatant from the FY strain compared to WT and the single mutant strains after overnight growth (representing the same final culture density; Fig S1b). The image is representative of three experiments. (d) The FY mutant is defective for secretion of AprE-FLAG. (e) The FY mutant is defective for secretion of heterologous AmyQ-His protein. For (d) and (e), strains were grown overnight to same cell density and centrifuged to obtain supernatant and pellet fractions. The levels of proteins were probed by immunoblotting with anti-FLAG or anti-His antibodies. Representative images of three independent experiments are shown. The full gels and Ponceau-stained images (to demonstrate equal loading) are shown in extended data Fig. S4. M, all stain precision blue marker (BioRad).
Figure 3.
Figure 3.. FY mutants are defective in LTA synthesis.
(a) Immunoblot detection of LTA with anti-LTA monoclonal antibodies. Note that in ltaS mutants the signal in the ~15–20 kDa range is absent, and instead longer polymers are detected that depend on the LtaSa enzyme. The lower inset shows an immunoblot for cells grown in LB + 50 μM Mn. The images are representative of two independent experiments with gels loaded with extracts from equal cell numbers. (b) Defects in LTA synthesis activate the sM-dependent envelope stress response as monitored using a luciferase transcriptional reporter fusion (PsigM-luxABCD). Cells were grown in LB broth with or without Mn (50 μM) or Ca (50 μM). Data is from three independent biological experiments and shown with mean ± SD. (c) Defective metalation of LTA synthase enzymes is associated with increased sensitivity to compound 1771, a LtaS inhibitor, . Aerobic growth of different strains (WT, FY, ltaS) in LB broth with or without 3 μM 1771 is shown. Data are representative of three independent cultures and presented as mean ± SD. Additional results, showing the effects of metal supplementation are in Fig. S9.
Figure 4.
Figure 4.. Complementation of the FY mutant with orthologous TerC proteins.
(a) Colony size (mean ± SD) of FY mutant with induction of TerC proteins on LB medium with 50 μM IPTG. TerC proteins were induced by 50 μM IPTG using Pspac(hy) promoter. Colony size was measured by imageJ. At least 40 isolated colonies measured for each strain. ****, P<0.0001, P value of each strain compared to FY samples was calculated using Welch’s t test, two-tailed. (b) Protease activities of FY mutant and FY complementary strains on 5% milk agar plates are shown. Cells were grown in LB broth with 50 μM IPTG inducer to OD600 0.4. Serial diluted cells (100 – 10−5) from these cultures were inoculated on the plates 37 °C for 24 hours.
Figure 5.
Figure 5.. The functions of TerC proteins MeeF and MeeY in exoenzyme metalation.
MeeF(F) and MeeY(Y) are integral membrane proteins that function in Mn export. F and Y are here shown exporting Mn to support metalation of exoenzymes. F and Y interact physically (coimmunoprecipitation) and genetically (epistasis with ftsH) with proteins of the secretosome complex. These results suggest that F and Y function co-translocationally to insert Mn into nascent metalloproteins. As a result, FY double mutants are deficient in Sec-dependent secretion of exoenzymes (e.g. proteases, AprE, AmyQ), which leads to growth defects on LB medium. FY mutants are also deficient in activation of LTA synthases, which bind Mn to an extracellular domain to catalyze lipoteichoic acid synthesis. The essentiality of FtsH in the FY mutant is consistent with jamming of the SecYEG translocon. F and Y may function as metallochaperones that directly transfer Mn to client proteins and/or they may help generate a sufficiently high local Mn concentration to allow metalation.
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