1. Secretion Monitor, SecM, Undergoes Self-Translation Arrest in the Cytosol 
Hitoshi Nakatogawa, Koreaki Ito 
Molecular Cell 
Volume 7, Issue 1, Pages (January 2001)
DOI: /S (01)

2. Figure 1 Synthesis and Stability of the secM Gene Products In Vivo
(A) SecM and SecM-Met6 synthesized in wild-type, secY-defective, and prc-defective cells. SecM (on pNH21; lanes 1–15) or SecM-Met6 (on pNH22; lanes 16–25) was induced for 30 min either in “wild-type” strain (GN40/pSTD343; lanes 1–5 and 16–20), secY24/Syd strain (Shimoike et al. 1995) (KI297/pST30; lanes 6–10 and 21–25), or prcΔ::neo strain (Hara et al. 1996) (JE7934; lanes 11–15) and subjected to pulse–chase and immunoprecipitation analyses. Note that Syd also was induced in the secY24/Syd strain, resulting in severely impaired SecY function (Matsuo et al. 1998). Band A represents the translation-arrested fragment. M and M′ represent the mature forms of SecM and SecM-Met6, respectively. P′ represents the full-length precursor form of SecM-Met6.
(B) The band A product lacks the C-terminal region. SecM-Met6 (on pNH22; lanes 1–5) and SecM-HA-Met6 (on pNH23; lanes 6–15) were analyzed by pulse–chase experiments. Samples were immunoprecipitated with either anti-SecM (lanes 1–10) or anti-HA (lanes 11–15). M′′ and P′′ indicate the products similar to M and P but retaining both Met6 and HA sequences.
Molecular Cell 2001 7, DOI: ( /S (01) )

3. Figure 2
Translation-Arrested and tRNA-Attached Forms of SecM Are Produced Both In Vivo and In Vitro
(A) CTABr fractionation of in vivo products. SecM-Met6 (on pNH22; lanes 1–8), SecM (on pNH21; lanes 9 and 10), SecM-HA-Met6 (on pNH32; lanes 11 and 12), and SS-HA-SecM-Met6 (on pNH31; lanes 13 and 14) were pulse labeled with [35S]methionine for 1 min and CTABr fractionated. Samples for lanes 3, 4, 7, and 8 were incubated with 100 μg/ml RNase A (at 37°C for 10 min) before the fractionation. Samples were subjected to SDS-PAGE either directly (lanes 1–4; sample sizes for lanes 2 and 4 were 1/10) or after immunoprecipitation (lanes 5–14). P and S indicate the pellet and the supernatant, respectively. A′′ indicates the translation-arrested product with the extra HA sequence after the signal sequence.
(B) Translation arrest in vitro. In vitro synthesis of SecM-Met6 was directed by pNH1 DNA (0.5 μg), in the presence of 45 μl of transcription-translation mixture (Baba et al. 1990), containing S140 extract from strain AD202 (Matsumoto et al. 1997), T7 RNA polymerase (45 units), and [35S]methionine. During incubation at 37°C, 10 μl samples were withdrawn for immunoprecipitation and SDS-PAGE (lanes 2–5). The 10 min sample was also CTABr fractionated (lanes 6 and 7). Lane 1 received an in vivo sample for comparison.
Molecular Cell 2001 7, DOI: ( /S (01) )

4. Figure 3 Defects in SecA and SRP Lead to Prolonged Translation Arrest
Cells were pulse labeled with [35S]methionine for 1 min and then chased for 0 (lanes 1 and 6), 1 (lanes 2 and 7), 2 (lanes 3 and 8), 4 (lanes 4 and 9), and 8 (lanes 5 and 10) min.
(A) SecM-Met6 was examined by pulse and chase in the presence (lanes 6–10) or the absence (lanes 1–5) of 0.02% sodium azide added 1 min before pulse labeling, followed by anti-SecM immunoprecipitation.
(B) SecM, under SRP-SRP receptor system-disturbed conditions, was examined using strain JM109(DE3) carrying pNH26 alone (lanes 1–5) or both pNH26 and pET9-FtsY (FtsY-overproducing plasmid; lanes 6–10) (Luirink et al. 1994).
(C) SecM-Met6 (on pNH5) was examined in strain WAM113 (Phillips and Silhavy 1992), in which ffh had been placed under the control of the araB promoter. Cells were grown first in M9 medium supplemented with 0.2% arabinose, washed three times with M9 salts, and inoculated (inoculum size, 1/500) into two portions of M9-amino acids media, one containing 0.2% arabinose (lanes 1–5 and A) and another containing 0.4% glucose (lanes 6–10 and G). After shaking at 37°C for 7 hr (with appropriate dilutions), SecM-Met6 was induced for 15 min and pulse–chase experiments were performed (lanes 1–10). Quantification of band A indicated that its half-life was 0.5 min for the arabinose-grown cells and 2 min for the Ffh-depleted cells. Ffh content was examined by immunoblotting (lanes A and G) using peptide antibodies against residues 419 to 432 of Ffh.
Molecular Cell 2001 7, DOI: ( /S (01) )

5. Figure 4
Defective as well as Deleted Signal Peptides Lead to Prolonged Translation Arrest
Cells were pulse labeled with [35S]methionine for 1 min and then chased for 0 (lanes 1 and 6), 1 (lanes 2 and 7), 2 (lanes 3 and 8), 4 (lanes 4 and 9), and 8 (lanes 5 and 10) min.
(A) ΔLGLPA-SecM-Met6 (on pNH30) was examined by pulse–chase and immunoprecipitation (lanes 6–12). The 0 min chase sample was CTABr fractionated (lanes P and S). Lanes 1–5 were for a control experiment using SecM-Met6. A* indicates the translation-arrested fragment of ΔLGLPA-SecM-Met6.
(B) ΔSS-′SecM-Met6 (on pNH7) was examined as in (A). A** indicates the translation-arrested fragment of ΔSS-′SecM-Met6.
Molecular Cell 2001 7, DOI: ( /S (01) )

6. Figure 5
Azetidine Incorporation Cancels the Enhanced Translation Arrest
Strain KI297/pST30 (secY24/Syd) harboring pNH22 (SecM-Met6) was grown in M9 medium supplemented with 17 amino acids (20 μg/ml each, other than Met, Cys, and Pro). SecM-Met6 and Syd were induced for 25 min, and then 100 μg/ml of either L-proline (lanes 1–4 and 9–12) or 2-azetidinecarboxylic acid (lanes 5–8 and 13–16) was added. Syd overproduction causes a translocation defect in the cells. Three minutes later, cells were subjected to pulse–chase and CTABr fractionation (lanes 1–8, precipitates; lanes 9–16, supernatant), followed by SecM immunoprecipitation.
Molecular Cell 2001 7, DOI: ( /S (01) )

7. Figure 6
Secretion Defect-Response of the Chromosomal secM-secA Expression
(A) Wild-type cells (KI298/pSTV29, Shimoike et al. 1995; lane 1) and the SecY-defective cells (KI297/pST30; lane 2) were induced with IPTG for 25 min and pulse labeled with [35S]methionine for 2 min. In the latter cells, Syd overproduction blocked the protein translocase. Samples were subjected to CTABr precipitation followed by SecM immunoprecipitation (bottom). They were also subjected to OmpA immunoprecipitation (middle) and SecA immunoprecipitation (top). Equal radioactivities were used between the two strains, but relative amounts used for CTABr precipitation, OmpA immunoprecipitation, and SecA immunoprecipitation were 60:1:2, respectively.
(B) Relative radioactivities associated with SecA (filled column) and the arrest fragment of SecM (open column) are compared between the two strains. Values for the wild-type strain were set as unity.
Molecular Cell 2001 7, DOI: ( /S (01) )