Intracellular localization of photosynthetic membrane growth initiation sites in Rhodopseudomonas sphaeroides

Abstract

Putative membrane invagination sites at which intracytoplasmic photosynthetic membrane growth is initiated in Rhodopseudomonas sphaeroides can be isolated in an upper pigmented fraction by rate-zone sedimentation. The intracellular localization of membranes present in the isolated fraction was investigated with the impermeant surface-labeling reagent pyridoxal 5'-phosphate, which has been shown to diffuse into the periplasmic space and to label proteins of both the peripheral cytoplasmic membrane and the mature intracytoplasmic membrane. A comparison of the extent of labeling at 25 and 0°C was consistent with the possibility that membranes present in the upper pigmented fraction arise from sites near the cell periphery. Pronase digestion of the surface-labeled membranes suggested further that the purified upper fraction consisted largely of open membrane fragments and that the majority of the intracytoplasmic membrane is labeled by this procedure. The pigmented membrane growth initiation sites were separated partially from undifferentiated respiratory cytoplasmic membrane also present in the upper fraction.

Membrane invagination in Rhodobacter sphaeroides is initiated at curved regions of the cytoplasmic membrane, then forms both budded and fully detached spherical vesicles

“…Therefore, ongoing protein synthesis is required to convert these UPB sites of incorporation of radiolabel to a more invaginated ICM structure. These results are in agreement with previous studies (Niederman et al, 1979;Inamine et al, 1984;Reilly and Niederman, 1986) and demonstrate that A. Separation into ICM and UPB membrane fractions by rate-zone ultracentrifugation on a 5-35% w/w sucrose gradient, following cell disruption.…”

“…1A). Pulse-chase radiolabelling with [S 35 ] methionine was carried out in order to align our experiments with earlier studies which demonstrated that the upper and lower membrane bands have a biosynthetic relationship, and originate from immature and mature membranes respectively (Niederman et al, 1979;Inamine et al, 1984;Reilly and Niederman, 1986). Figure 1B shows that a short pulse of radiolabel is incorporated almost instantaneously into the UPB membrane, with a subsequent decline over the 250 min time-course of the chase with unlabelled methionine.…”

“…interconnected ICM vesicles, or from vesicles completely detached from any other cellular structure. The rate-zone sucrose density gradients used in this and previous work by Niederman and colleagues (Parks and Niederman, 1978;Niederman et al, 1979;Inamine et al, 1984;Reilly and Niederman, 1986) separate structures based on the rate at which they sediment rather than their density, so the size and shape of the membranes influence the banding pattern. The two major membrane morphologies, open patches (UPB) and spheres (ICM; see Fig.…”

“…When Rba. sphaeroides cells are disrupted by passage through the French press cell the shear forces break the continuity of the cell membrane and three regions, the unpigmented CM, the ICM, and the ICM precursor, the UPB, can all be isolated from cell extracts (Parks and Niederman, 1978;Niederman et al, 1979;Inamine et al, 1984;Reilly and Niederman, 1986). Apparently, cell disruption and subsequent purification severs the curved UPB membranes from the surrounding CM.…”

PucC and LhaA direct efficient assembly of the light‐harvesting complexes in Rhodobacter sphaeroides

“…Figure B and E compares the CN‐PAGE analyses of mature intracytoplasmic membranes (ICM) and the sites of initiation of membrane invagination [upper pigmented band (UPB)] that are enriched in proteins involved in pigment and protein biogenesis (Niederman et al ., ; Woronowicz and Niederman, ; Jackson et al ., ). The sample loadings were balanced for protein content, as seen in the Coomassie stained lanes, so there is far less pigment in the corresponding unstained UPB lanes, reflecting their cellular origin as indentations of the non‐photosynthetic cytoplasmic membrane (Inamine et al ., ; Tucker et al ., ). Figure B,E analyse the FLAG‐LhaA and FLAG‐PucC strain, respectively; Fig.…”