Fig. 2.
Optimized lysine racemase for cell-specific labeling with D-lysine. A, Proteus mirabilis lysine racemase (Lyr) constructs. Wild-type Lyr (LyrWT) contains a putative signal peptide (amino acids 1–36) (green) and a globular catalytic domain (amino acids 37–407) (light blue). Catalytic residues K74 and Y299 are highlighted (blue). To limit secretion of Lyr, constructs were prepared without the signal peptide (LyrM37) and with a KDEL endoplasmic retention motif (red) (LyrM37-KDEL). B, C3H10T1/2 proliferation on titrated concentrations of D-lysine when transfected with an empty vector, LyrWT, LyrM37, or LyrM37-KDEL. Results are expressed as a percentage of cells grown in 10 mm L-lysine. C, full-length LyrWT was readily secreted from cells (solid arrow), whereas cleaved LyrWT remained intracellular (hollow arrow). LyrM37 limited extracellular Lyr, and the addition of a KDEL motif ablated Lyr secretion. D, LyrWT located to the ER (for secretion), LyrM37 was spread throughout the cell, and LyrM37-KDEL resided within the ER (for retention). E, stable C3H10T1/2+LyrM37-KDEL cells were grown on titrated concentrations of L-lysine, D-lysine, or meso-2,6-diaminopimelate (DAP). LyrM37-KDEL conferred cell proliferation comparable to L-lysine when the D-lysine concentration was >1 mm. LyrM37-KDEL cells did not grow on DAP. F, C3H10T1/2+LyrM37-KDEL cells were grown for 24 h in 10 mm L-lysine, the absence of lysine, or titrated concentrations of D-lysine. The transcriptional arrest marker pS51 eIF2α was measured by Western blotting. Concentrations of D-lysine greater than 1 mm rescued the amino acid starvation response. G, isotopic labeling efficiency of C3H10T1/2+LyrM37-KDEL cells grown on 2.5 mm light, medium (+4 Da), or heavy (+8 Da) L-lysine (i.e. SILAC) and D-lysine (five passages). Each data point represents an individual peptide, and mean labeling percentages are indicated by a red line. Error bars = standard deviation.