Isolation and Characterization of the Amyloplast Envelope-Membrane from Cultured White-Wild Cells of Sycamore (Acer pseudoplatanus L.)

Abstract

To study the characteristic features of the amyloplast, a uniquely differentiated plastid-type which synthesizes and accumulates reserve starch, in comparison with those of the chloroplast, these two types of plastids were isolated from white-wild and green-mutant protoplasts of cultured sycamore (Acer pseudoplatanus L.) cells, respectively. The intactness of the isolated amyloplast preparations was 70%. Electron microscopic ultrastructural analysis of both plastid types revealed unique structural features of the green-mutant chloroplasts, including well developed grana membranes and abundant ribosomal particles and plastoglobuli. After osmotic rupture of the isolated amyloplasts and chloroplasts, a clear separation of the envelope-membranes was achieved by discontinuous sucrose density gradient centrifugation. Although the visible absorption spectra of the envelope lipid components were indistinguishable between the amyloplasts and chloroplasts, the envelope-membrane polypeptide patterns were clearly distinct as judged by denaturing electrophoresis. By immunoblotting analysis using the specific antiserum raised against the pea chloroplast 29-kilodalton Pi-translocator, the amount of this carrier-protein (31-kilodalton) in the white-wild amyloplast envelope-membranes was estimated to be at least 10-fold less than in the green-mutant envelopes.The amyloplast is a uniquely differentiated plastid-type which synthesizes and accumulates starch in the stromal matrix. It has long been hypothesized that this organelle is ontogenically and I

Immunochemical Analysis Shows That an ATP/ADP-Translocator Is Associated with the Inner-Envelope Membranes of Amyloplasts from Acer pseudoplatanus L.

“…The profiles visualized by silver staining are quite different between them (lanes 3 and 6), and densitometric tracing clearly indicates that there are specific bands characteristic of each inner-membrane fraction (data not shown). It should be noted that, consistent with our previous reports (8,9,16), there is a major polypeptide (32-kD) (closed circle) in the amyloplast inner-membrane which is clearly separable from a faint band of 31-kD band corresponding to the Pitranslocator (see below and Fig. 3) (star), whereas the 3 l-kD protein is a dominant molecular species in the inner-membrane of the chloroplasts.…”

“…Culture methods and the isolation of intact amyloplasts and chloroplasts from white-wild and green-mutant cells of sycamore (Acer pseudoplatanus L.), respectively, were exactly the same as those reported previously (16,17). In addition, the specific mitochondrial marker-enzyme Cyt cl has been examined in the plastid preparations as well.…”

“…Throughout this investigation related to the biochemistry of the amyloplasts, we have used functional chloroplasts isolated from the green-mutant cell-line of sycamore as a critical control system (16,17).…”

“…In both cases, the Pitranslocator in the inner-envelope membranes is thought to play a crucial role (16), although it remains unclear whether its function in the amyloplasts is truly identical to that operating in the chloroplast envelope. Another key component which possibly resides in the amyloplast inner-envelope is a putative ATP/ADP-translocator (16); since this plastid type lacks the energy-producing machinery associated with the thylakoid membranes in chloroplasts, the ATP which is required for the progress of gluconeogenesis (i.e. ADP-glucose formation) must be imported from the cytosol.…”

“…Based on these reasons, we view the chloroplast and amyloplast each as a "'source," "sink," and "gluconeogenic" organelle (16 …”