G. obscuriglobus cells uptake proteins but not DNA in an energy-dependent process. (A) Bright field microscopy and CLSM images of G. obscuriglobus cells incubated with GFP (10 μg/mL) for 60 min. Left and Right microscopy images correspond to bright field and GFP fluorescence, respectively. Because of optical sectioning effects, only some of the cells in the clump positive for GFP signal can be visualized. (Scale bar, 4 μm.) (B) Time-course experiment with GFP alone (filled circles), with GFP and 1 mM sodium azide (open circles), or with GFP and both 1 mM sodium azide and 5 mM ATP (filled triangles). After washing of G. obscuriglobus cells, GFP fluorescence was quantitatively analyzed by microplate reader assay. Error bars represent the SD from three experimental replicates. (C) Western blot analysis showing uptake of full-length GFP (≈27 kDa) by G. obscuriglobus. Cells were incubated with GFP for 1 h. After washing, cell lysates were electrophoretically separated on an SDS gel (Left), then blotted and probed with an anti-GFP antibody (Right). Lane 1, E. coli DH5α incubated with GFP; lane 2, G. obscuriglobus incubated without GFP; lane 3, G. obscuriglobus incubated with GFP. (D) Competition uptake experiment with GFP, together with either BSA or DNA. G. obscuriglobus cells were incubated for 1 h with 10 μg/mL of GFP alone, with GFP and BSA (100 μg/mL), or with GFP and plasmid DNA (100 μg/mL). Cells were then washed and analyzed for fluorescence emission by microplate reader assay. Error bars represent the SD from three experimental replicates.

Internalized proteins in G. obscuriglobus cells are localized to paryphoplasm. (A) G. obscuriglobus cells were incubated with GFP and then stained with DAPI and SynaptoRed. A GFP-containing region is seen in the cytoplasm bounded by the cytoplasmic membrane as defined by the SynaptoRed staining and is separated from the nuclear body (DAPI staining). (B) TEM image of a section of high-pressure frozen cryosubstituted cells of G. obscuriglobus, immunogold-labeled to detect GFP via anti-GFP antibody and secondary antibody conjugated with 10 nm colloidal gold. Gold particles (short arrows) labeling internalized GFP are only associated with paryphoplasm (P). Gold particles were excluded from both the double membrane-bounded nucleoid and the riboplasm. The riboplasm (R), fibrillar nucleoid (N), and intracytoplasmic membrane (ICM) are indicated. (Scale bar, 500 nm.) (C) Diagram representing the functional compartmentalization of G. obscuriglobus. N, nucleoid; NE, nuclear envelope; ICM, intracytoplasmic membrane; R, riboplasm; CM, cytoplasmic membrane; CW, cell wall.

TEM of high-pressure frozen cryosubstituted G. obscuriglobus cells showing association of GFP and MC protein with membranes and vesicles. (A) Cells were immunogold-labeled to detect GFP, which is seen only in the paryphoplasm. A1 and the enlargements of boxed areas 2–4 show association of several gold particles with the same membrane. Arrowheads, membranes; V, vesicles. (Scale bars, 1 μm in A1, 50 nm in A2–A4.) (B) A section showing infolding of cytoplasmic membrane (arrowheads) associated with GFP (gold particles). (Scale bar, 50 nm.) (C) Cells were incubated with HRP-conjugated rabbit anti-mouse antibody. Peroxidase activity was detected via cytochemistry using diaminobenzidine substrate, yielding an electron-dense product. An example is shown of a region where the cytoplasmic membrane (CM) seems to have invaginated (arrowheads) together with HRP-conjugated antibody, as indicated by the dense reaction product. (Scale bar, 50 nm.) (D) G. obscuriglobus cells preincubated with GFP were immunogold-labeled to detect MC protein. D1 and enlargements of boxed areas 2 and 3 show association of several gold particles with membrane vesicle. Arrowheads, membranes; V, vesicles. (Scale bars, 500 nm in D1, 200 nm in D2, 50 nm in D3.)

Colocalization of GFP and MC proteins to the same density gradient membrane fractions of lysed G. obscurigobus cells. These cells had been incubated with GFP. (A and B) Immunogold labeling of the material obtained from fraction 9 of the 40–70% sucrose density gradient centrifugation (Fig. S9) showing cofractionation of GFP and MC protein and association of both proteins with membranes. (A) Fraction 9 was immunolabeled with anti-GFP antibody and secondary anti-mouse-10 nm gold antibody to visualize GFP. Gold particles (g) indicate that GFP is associated with broken membranes (Upper) and the vesicle membrane (Lower) rather than the vesicle interior. Arrowheads, membranes; V, vesicle. (Scale bar, 200 nm.) (B) Fraction 9 was immunolabeled with anti-gp4978 antibody and protein A-15 nm gold to visualize MC-like gp4978 protein. Arrowheads (membranes) and gold particles (g) indicate that MC protein is associated with broken membranes. (Scale bar, 200 nm.)