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

Abstract

Pure preparations of intact amyloplasts and chloroplasts, free from mitochondrial contamination, were isolated from cultured cells of the white-wild and green-mutant lines of sycamore (Acer pseudoplatanus L.), respectively. A specific rabbit antiserum against yeast mitochondrial cytochrome c1 only cross-reacted with mitochondrial membranes from the white-wild sycamore cells. The outer and inner envelope-membranes of the two plastid-types were isolated and subsequently analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis to characterize polypeptide pattems in each fraction. Analysis by immunoblotting clearly showed that antiserum against the 29-kilodalton inorganic orthophosphate translocator isolated from pea chloroplasts cross-reacted with a 31-kilodalton polypeptide residing in the inner-envelope membranes from both sycamore chloroplasts and amyloplasts. In contrast, antiserum against the ADP/ATPtranslocator isolated from mitochondria of Neurospora crassa yielded a positive signal with a 32-kilodalton polypeptide in the inner-membranes isolated from amyloplasts, but not green-mutant chloroplasts. We propose that this 32-kilodalton polypeptide in the amyloplast envelope is a putative ATP/ADP-translocator and its possible functional significance is discussed.assimilates (triose-P) to the cytosol via the Pi-translocator, carbon-partitioning in amyloplasts will be directed to the uptake ofcarbon-compounds from the cytosol into the stroma and their eventual conversion to starch. 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.To address the question of whether or not an ATP/ADPtranslocator is specifically present in the inner membrane of the amyloplast envelope, we have exploited immunoblot analysis using antiserum against the ADP/ATP-translocator isolated from the mitochondrial membranes of Neurospora crassa (22). 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).The non-green amyloplast, a uniquely differentiated plastid-type which synthesizes and accumulates starch in the stromal matrix, possesses a unidirectional gluconeogenic pathway in contrast to the vast functional diversity of the chloroplast, including energy (ATP) formation, photosynthetic C02-fixation and gluconeogenesis among several others. Based on these reasons, we view the chloroplast and amyloplast each as a "'source," "sink," a...

Starch biosynthesis.

“…This enzyme is active within the plastid, which means that its substrates, glucose-1-phosphate and ATP, must also be present in the plastid. In chloroplasts, ATP may be derived from photosynthesis, but in nonphotosynthetic plastids, it must be specifically imported from the cytosol, probably by an ADP/ATP translocator (Ngernprasirtsiri et al, 1989;Schünemann et al, 1993). The glucose-1-phosphate can be supplied by the reductive pentose phosphate pathway in chloroplasts via phosphoglucoisomerase and phosphoglucomutase .…”

Direct transport of ADPglucose by an adenylate translocator is linked to starch biosynthesis in amyloplasts.

“…Previously, we reported the detection, by immunoanalysis, of a putative adenylate (ATP/ADP) translocator in the amyloplast envelopes isolated from cultured sycamore cells (6). We have now examined the kinetic properties of this transporter by using intact amyloplast preparations and have found evidence for the direct transport of ADP-Glc across the amyloplast envelope, which is tightly linked to the formation of starch.…”

Molecular characterization of an Arabidopsis thaliana cDNA encoding a novel putative adenylate translocator of higher plants

“…However, Schtinemann et al [20] failed to show any cross-reactivity of the same antibody with proteins in the envelope membrane of pea root plastids and could not reproduce the results obtained by Pozueta-Romero et al [29] with spinach chloroplast envelope membranes. Moreover, the same group did not find a cross-reaction with proteins from sycamore chloroplast envelope membranes [31]. Thus, at present it seems more than questionable that the adenylate translocators in plastids and mitochondria are homologous membrane proteins.…”