Protein localization and dynamics within a bacterial organelle

Abstract

Protein localization mechanisms dictate the functional and structural specialization of cells. Of the four polar surface organelles featured by the dimorphic bacterium Caulobacter crescentus, the stalk, a cylindrical extension of all cell envelope layers, is the least well characterized at the molecular level. Here we apply a powerful experimental scheme that integrates genetics with high-throughput localization to discover StpX, an uncharacterized bitopic membrane protein that modulates stalk elongation and is sequestered to the stalk. In stalkless mutants StpX is dispersed. Two populations of StpX were discernible within the stalk with different mobilities: an immobile one near the stalk base and a mobile one near the stalk tip. Molecular anatomy provides evidence that (i) the StpX transmembrane domain enables access to the stalk organelle, (ii) the N-terminal periplasmic domain mediates retention in the stalk, and (iii) the C-terminal cytoplasmic domain enhances diffusion within the stalk. Moreover, the accumulation of StpX and an N-terminally truncated isoform is differentially coordinated with the cell cycle. Thus, at the submicron scale the localization and the mobility of a protein are precisely regulated in space and time and are important for the correct organization of a subcellular compartment or organelle such as the stalk.

Fig. 1.

Localization of StpX-GFP in wild-type (WT) and mutant cells that are stalkless or that have elongated stalks in which WT stpX is replaced by stpX-GFP under the control of the native promoter. (A–E) Colocalization of CC1679-mCherry and StpX-GFP in NA1000 (A–C) or CB15 cells (D and E). Cells harbor stpX-GFP and pP_van-CC1679-mCherry, a low-copy plasmid encoding CC1679-mCherry under the control of the vanillate-inducible promoter (P_van). (F–N and P) Mutants harboring stpX-GFP. (L and N) StpX-GFP cells with pP_xyl-StaR, a low-copy plasmid encoding StaR under the control of the xylose-inducible promoter (P_xyl). (O) Time-course fluorescence imaging of stpX-GFP cells during cell-cycle progression. Numbers in DIC panels indicate the time in minutes samples were imaged, relative to the start of the cell cycle (SW cell stage). A summarizing schematic shown beneath the images depicts delocalized (light green) and localized (bright green) StpX-GFP in SW and ST cells, respectively. (P) Localization of StpX-GFP after exposure of stpX-GFP SW cells to A22 (12.5 μg/mL) to inhibit stalk outgrowth. Addition of the same amount of solvent (methanol) had no effect on StpX-GFP localization.

Fig. 2.

Stalk elongation control by StpX. (A) Stalk length distribution in NA1000 WT cells containing either the control vector (pMT375) or pP_xyl-StpX, a low-copy plasmid encoding StpX under P_xyl control, after overnight growth in PYEX (PYE supplemented with 0.3% xylose). (B) Stalk length distribution in ΔphoB single mutants and ΔphoB ΔstpX double mutants grown in PYE. (C) Stalk length distribution of NA1000 WT and ΔstpX cells grown in phosphate-limiting conditions (HIGG medium + 30 μM phosphate).

Fig. 3.

Localization determinants and accumulation of StpX isoforms. (A) Schematic of the predicted primary structure predictions of StpX, StpX-GFP, and deletion derivatives, including the signal sequence peptide (SS, gray box), the periplasmic domain (per, red box), the transmembrane (TM) domain (yellow box), the cytoplasmic domain (cd, turquoise box), and the GFP moiety (green box). The numbers refer to amino acid residues of StpX. (B) Fluorescence image (3s, 3-sec exposure) analyses of cells expressing StpX(Δper)-GFP or StpX(ΔperΔtm)-GFP from the native chromosomal locus under control of its endogenous promoter in place of WT StpX. Yellow arrowheads point to fluorescent stalks. (C) Immunoblot analysis of StpX-GFP at different stages of the cell cycle (see schematic in Fig. 10; 20-min intervals indicated by multiples of 20), using polyclonal antibodies to GFP (α-GFP). (D) Immunoblot analysis of NA1000 (control), stpX-GFP, stpX(Δcd)-GFP, and the strains in B, using a polyclonal antibody to GFP. StpX-GFP, StpX(Δper)-sEGFP, StpX(ΔperΔtm)-GFP, and StpX(Δcd)-GFP are abbreviated as X-GFP, XΔP-GFP, XΔPT-GFP, and XΔCD-GFP, respectively. (E) Immunoblots of endogenous (untagged) StpX in WT and deletion strains using polyclonal antibodies to the C-terminal domain of StpX. (C–E) Full-length (red asterisk) and truncated (blue asterisk) StpX (E) or StpX-GFP (C and D) are indicated. (C and E) Immunoblots of CtrA are shown as a control for the cell cycle and/or loading. (F) NA1000 cells expressing a short polypeptide with (Left) or without (Center) the TM domain of StpX or that from the bitopic membrane protein PflI (CC2060, Right) fused to GFP under P_van control from plasmids. Cells were imaged after 5 h of growth in the presence of 0.5 mM vanillate. Cells harbor pCWR435, pCWR437, and p2060-TM-long-510 encoding the short polypeptide-GFP chimeras TM-GFP, ΔTM-GFP, and TM-GFP that derive from StpX(Δper), StpX(ΔperΔtm), and PflI, respectively (labeled beneath the images as “origin”).

Fig. 4.

Mobility of StpX-GFP and mutant derivatives in vivo as determined by FLIP and/or FRAP analysis in HIGG (30 μM phosphate). (A–E) FLIP analysis of cells expressing StpX-GFP (A–C; “WT”), StpX(Δper)-GFP (A and D–E; “Δper”) and StpX(Δcd)-GFP (A; “Δcd”). Fluorescence images were acquired before (“pre”) and after (“post”) bleaching (52 sec) the area defined by the white boxes. Yellow arrowheads denote the loss in fluorescence (bleaching) in areas of the stalks that were not directly illuminated by the laser. Red arrowheads denote cell bodies. (F) FRAP analysis of cells expressing StpX(Δper)-GFP (“Δper”). Images were captured before (“pre”) and immediately after (“post”) bleaching (13 sec) the white boxed area and finally again after 96 sec of recovery (“recovery”). A Left shows the quantification of fluorescence signals of the images in the Right before (“pre-bleach”, blue line) and after (“post-bleach”, red line) illumination with the laser. The gray sections denote the bleached regions. The extent of fluorescence loss in regions adjacent to bleached regions is indicated above the relevant areas by arrays of triangles.