Soluble protein kinase and protein phosphatase activities were localized in the cytosol of pea mesophyll cells using protoplasts fractionation techniques. The molecular weights of the phosphorylated cytosolic proteins, as determined by polyacrylamide gel electrophoresis, were 68, 55, 46, 38, 36, 30, 22 and 12 kDa. Histone and, to a much lesser extent, casein but not phosvitin were accepted as exogenous substrates. In every case serine served as acceptor amino acid for the phosphate residue. The protein phosphorylation activity had an alkaline pH optimum, and showed no response to varying Mg-2, Ca2+, Pi cyclo-AMP or calmodulin concentrations. The kinase activity was competitively inhibited by ADP and pyrophosphate with apparent Ki values of 0.5 and 0.17 mᴍ , respectively. High ATP concentrations (1-4 mᴍ) resulted in a strong decrease of radioactivity in the 32P labeled proteins. It is proposed that the ratio of protein phosphorylation to protein dephosphorylation is regulated by the ATP to ADP ratio in the cytosol.
Plastids originated from an endosymbiotic event between
an early eukaryotic host cell and an ancestor of
today's cyanobacteria. During the events by which the
engulfed endosymbiont was transformed into a permanent
organelle, many genes were transferred from
the plastidal genome to the nucleus of the host cell.
Proteins encoded by these genes are synthesised in
the cytosol and subsequently translocated into the
plastid. Therefore they contain an N-terminal cleavable
transit sequence that is necessary for translocation.
The sequence is plastid-specific, thus preventing
mistargeting into other organelles. Receptors embedded
into the outer envelope of the plastid recognise
the transit sequences, and precursor proteins are
translocated into the chloroplast by a proteinaceous
import machinery located in both the outer and inner
envelopes. Inside the stroma the transit sequences
are cleaved off and the proteins are further routed to
their final locations within the plastid.
The energy requirement for protein transport into chloroplast was assayed under conditions that perm it to distinguish whether the posttranslational translocation is dependent on ATP or whether a membrane potential across the chloroplast envelope can drive this transport event.
A membrane potential is not required for translocation. ATP can support protein transport in the presence of protonophores and ionophores. Non-hydrolyzable ATP analogues and GTP, CTP, UTP cannot serve as ATP substitutes. Translocation could be observed when an ATP generating system was used to supply ATP. In contrast ATP degrading systems completely abolished translocation.
The inner envelope mem brane localized ATP-ase is probably not involved in the transport event. The results suggest that ATP is needed at the outer chloroplast envelope.
Inhibition of protein transport by ADP, pyrophosphate and NaF is studied and its consequences discussed.
Chloroplast differentiation in angiosperm plants depends
on the light-dependent conversion of protochlorophyllide
to chlorophyllide by NADPH:protochlorophyllide
oxidoreductase (PORA; EC 188.8.131.52), a
nuclearly encoded protein. The protein import of the
precursor form of PORA into plastids was shown
previously to strictly depend on the presence of
its substrate protochlorophyllide. PORA seemed to
follow a novel, posttranslationally regulated import
route. Here we demonstrate that the precursor of
PORA from barley is imported into isolated barley
plastids independently of protochlorophyllide. PORA
as well as PORB import is competed for by the precursor
of the small subunit of Rubisco. The data demonstrate
that the PORA precursor uses the general
import pathway into plastids. Furthermore, en route
into chloroplasts the pea POR precursor can be crosslinked
to the protein import channel in the outer envelope
Toc75 from pea.
Protein import into plant chloroplasts is a fascinating topic that is being investigated by many research groups. Since the majority of chloroplast proteins are synthesised as precursor proteins in the cytosol, they have to be posttranslationally imported into the organelle. For this purpose, most preproteins are synthesised with an N-terminal presequence, which is both necessary and sufficient for organelle recognition and translocation initiation. The import of preproteins is facilitated by two translocation machineries in the outer and inner envelope of chloroplasts, the Toc and Tic complexes, respectively. Translocation of precursor proteins across the envelope membrane has to be highly regulated to react to the metabolic requirements of the organelle. The aim of this review is to summarise the events that take place at the translocation machineries that are known so far. In addition, we focus in particular on alternative import pathways and the aspect of regulation of protein transport at the outer and inner envelope membrane.
Chloroplasts, unique organelles of plants, originated from endosymbiosis of an ancestor of today's cyanobacteria with a mitochondria-containing host cell. It is assumed that the outer envelope membrane, which delimits the chloroplast from the surrounding cytosol, was thus inherited from its Gram-negative bacterial ancestor. This plastid-specific membrane is thus equipped with elements of prokaryotic and eukaryotic origin. In particular, the membrane-intrinsic outer envelope proteins (OEPs) form solute channels with properties reminiscent of porins and channels in the bacterial outer membrane. OEP channels are characterised by distinct specificities for metabolites and a quite peculiar expression pattern in specialised plant organs and plastids, thus disproving the assumption that the outer envelope is a non-specific molecular sieve. The same is true for the outer membrane of Gram-negative bacteria, which functions as a permeability barrier in addition to the cytoplasmic membrane, and embeds different classes of channel pores. The channels of these prokaryotic prototype proteins, ranging from unspecific porins to specific channels to ligand-gated receptors, are exclusively built of β-barrels. Although most of the OEP channels are formed by β-strands as well, phylogeny based on sequence homology alone is not feasible. Thus, the comparison of structural and functional properties of chloroplast outer envelope and bacterial outer membrane channels is required to pinpoint the ancestral OEP ‘portrait gallery’.
Chloroplast biogenesis often requires a tight orchestration between gene expression (both plastidial and nuclear) and translocation of ∼3000 nuclear-encoded proteins into the organelle. Protein translocation is achieved via two multimeric import machineries at the outer (TOC) and inner (TIC) envelope of chloroplast, respectively. Three components constitute the core element of the TOC complex: a β-barrel protein translocation channel Toc75 and two receptor constituents, Toc159 and Toc34. A diverse set of distinct TOC complexes have recently been characterized and these diversified TOC complexes have evolved to coordinate the translocation of differentially expressed proteins. This review aims to describe the recent discoveries relating to the typical characteristics of these distinct TOC complexes, particularly the receptor constituents, which are the main contributors for TOC complex diversification.