Kinetics of 125I-ubiquitin conjugation with and liberation from rabbit reticulocyte stroma

The breakdown of mitochondria-containing stroma of rabbit reticulocytes is an ATP- and ubiquitin-dependent process and there is no evidence for an ATP-dependent but ubiquitin-independent proteolysis in these cells. The ubiquitin conjugate formation with heat-denatured stroma proteins is about one-fifth of that with native stroma. In reticulocytes there exist two mechanisms of ubiquitin liberation from its conjugates with stroma proteins: an ATP-dependent and hemin-resistant release of ubiquitin, which is assumed to be the first step in the degradation of ubiquitin conjugates by the protease system, and a release of ubiquitin catalyzed by an isopeptidase activity.     

Turnover of excess hemoglobin alpha chains in beta-thalassemic cells is ATP-dependent

Blood erythroid cells from five beta-thalassemic donors were incubated with [3H]leucine at 37 degrees C to label the pool of excess, free hemoglobin alpha chains. The 3H-labeled cells were split and reincubated in nonradioactive media that either supported ATP production (with 5 mM glucose) or inhibited ATP production (without glucose but with 20 mM 2-deoxyglucose and 0.20 mM 2,4-dinitrophenol). During the 6-h incubation in the glucose medium, the total cellular protein 3H radioactivity decreased about 30%, while the ATP levels remained constant. Chromatographic separation of the alpha- and non-alpha-globin chains of the crude (stroma included) lysates and electrophoretic separation of the free alpha chains and tetrameric hemoglobins of the stroma-free soluble phases both showed that degradation of the alpha chains was responsible for the decrease in protein 3H radioactivity. Conversely, in the energy-deprived cells, the ATP levels dropped to less than 10% of that of the energy-supported cells, and the turnover of alpha-globin 3H radioactivity of the crude lysates was only 5-10%. These results indicate that proteolysis of excess, newly synthesized alpha chains in beta-thalassemic cells is ATP-dependent. The accumulation, mostly in the stromal fraction, of intact 3H-alpha chains in the ATP-deprived cells suggests that an ATP-dependent step occurs early in the biochemical pathway of alpha chain proteolysis. Denaturation resulting in insolubility of the free alpha chains may be a recognition signal for activation of this proteolysis.     

Functional changes of mouse spermatozoa stored in vitro

In suspensions of epididymal spermatozoa in vitro at +10°C and +37°C, all nuclei-containing and mitochondria-containing structures (normal spermatozoa, spermatozoa with the bent and coiled tails, complexes of head and neck) are with propidium iodide and rhodamine 123, respectively. Intracellular ATP concentration determined by a bioluminescent method in mitochondria-containing elements of suspension decreases (significantly faster at 37°C than at 10°C) up to a certain unchangeable level (2.5 × 10^?8 M/l at 37°C and to 1.6 × 10^?8 M/l at 10°C per 10⁶ of mitochondria-containing elements). Mechanisms of spermatozoa destruction are discussed.     

ATP-dependent peptide release from mitochondria of reticulocytes

ATP-dependent release of TCA-precipitable peptides from mitochondria-containing stroma (MCS) is described. The process is independent of ubiquitin, but is sensitive to hemin and to heat treatment. Neither chloramphenicol nor EGTA inhibit. 50% of the activity is dependent on charged tRNA. The peptides released from MCS possess a molecular mass of about 1–5 kDa and are degraded to TCA-soluble compounds by a cytosolic protease system (fraction II) without ubiquitin.     

Further evidence for a proton pump in mouse kidney phagolysosomes: Effect of nigericin and 2,4-dinitrophenol on the stimulation of intralysosomal proteolysis by ATP

The lipid soluble acid 2,4-dinitrophenol completely abolished the stimulatory effect of ATP on intralysosomal proteolysis in mouse kidney phagolysosomes at pH 8. The protonophore had no effect in the absence of ATP at pH 8 but inhibited intralysosomal proteolysis in unbuffered media. The ionophorous antibiotic nigericin also prevented the action of ATP at pH 8 but had no effect in the absence of ATP in unbuffered media. Nigericin also inhibited intralysosomal proteolysis at pH 8 in the absence of ATP. These observations support the hypothesis that the phagolysosome membrane contains an ATP-driven proton pump which functions to maintain intralysosomal acidity.     

Maize non-photosynthetic ferredoxin precursor is mis-sorted to the intermembrane space of chloroplasts in the presence of light

Preprotein translocation across the outer and inner envelope membranes of chloroplasts is an energy-dependent process requiring ATP hydrolysis. Several precursor proteins analyzed so far have been found to be imported into isolated chloroplasts equally well in the dark in the presence of ATP as in the light where ATP is supplied by photophosphorylation in the chloroplasts themselves. We demonstrate here that precursors of two maize (Zea mays L. cv Golden Cross Bantam) ferredoxin isoproteins, pFdI and pFdIII, show distinct characteristics of import into maize chloroplasts. pFdI, a photosynthetic ferredoxin precursor, was efficiently imported into the stroma of isolated maize chloroplasts both in the light and in the dark. In contrast pFdIII, a non-photosynthetic ferredoxin precursor, was mostly mis-sorted to the intermembrane space of chloroplastic envelopes as an unprocessed precursor form in the light but was efficiently imported into the stroma and processed to its mature form in the dark. The mis-sorted pFdIII, which accumulated in the intermembrane space in the light, could not undergo subsequent import into the stroma in the dark, even in the presence of ATP. However, when the mis-sorted pFdIII was recovered and used for a separate import reaction, pFdIII was capable of import into the chloroplasts in the dark. pFNRII, a ferredoxin-NADP+ reductase isoprotein precursor, showed import characteristics similar to those of pFdIII. Moreover, pFdIII exhibited similar import characteristics with chloroplasts isolated from wheat (Pennisetum americanum) and pea (Pisum sativum cv Alaska). These findings suggest that the translocation of precursor proteins across the envelope membranes of chloroplasts may involve substrate-dependent light-regulated mechanisms.     

ATP activation of protein degradation by extracts of crude and purified lysosomal preparations

Activation of proteolysis by ATP was studied in lysates of crude and purified lysosomal preparations from liver and kidney at acid pH. In the crude system, from kidney, it was found that ATP activates proteolysis over a concentration range of 0.1-2 mM. Up to 4-fold activation was observed. GTP and CTP also activated proteolysis, but to a lesser extent. Proteolysis was inhibited by vanadate and molybdate. Fractionation of the kidney lysosomes on Percoll gradients produced two fractions containing lysosomal marker enzymes. Most of the acid phosphatase and the acid pyrophosphatase were found in the lighter band, while most of the beta-galactosidase and cathepsin activity was found in a more dense band. Proteolysis by lysates of both fractions was activated by ATP and inhibited by vanadate and molybdate. In the dense band proteolysis was also nearly totally blocked by pepstatin, and was enhanced by an inhibitor of pyrophosphatases, sodium fluoride. ATP also activates proteolysis in crude lysosomes from liver, but upon fractionation of this tissue it was found that all the lysosomal enzyme markers are present in the dense fraction obtained from the Percoll gradient. Again, proteolysis by lysates of the purified fractions was activated by ATP and inhibited by vanadate and molybdate. These data indicate that ATP can activate proteolysis at acid pH in a lysosomal milieu containing enzymes which also catalyze its breakdown. In the kidney there may be two lysosomal compartments which separate the enzymes catalyzing ATP breakdown from the proteolytic enzymes, but this is not essential for ATP activation as shown by the data from the liver and the crude lysosomal fractions.     

ATP-dependent proteolysis of hemoglobin alpha chains in beta-thalassemic hemolysates is ubiquitin-dependent

beta-Thalassemia is an inherited human disorder which is characterized by a deficient production of hemoglobin beta chains and an attendant accumulation of structurally normal alpha chains in the erythropoietic cells. The objective of this work is to understand the mechanism of intracellular proteolysis of these excess alpha chains. Dialyzed stroma-free hemolysates (32 mg/ml hemoglobin) of blood reticulocytes from four individuals with beta-thalassemia intermedia were incubated with human hemoglobin 3H-alpha chains (0.13 mg/ml) at 37 degrees C in a reaction mixture supporting protein degradation. In the presence of ATP and an ATP-generating system, the fraction of alpha chain 3H radioactivity made acid-soluble after 4 h ranged from 4 to 12% among the different hemolysates; in the absence of ATP or when hemolysates of normal human erythrocytes were used, only 1 to 2% of the 3H-alpha chains were degraded. It is likely that the ATP-dependent proteolysis of 3H-alpha chains in the beta-thalassemic hemolysates corresponds to the ATP-dependent turnover of newly synthesized soluble alpha chains in intact beta-thalassemic reticulocytes observed previously (Shaeffer, J. (1983) J. Biol. Chem. 258, 13172-13177) because of the following similarities between the two systems: (a) free 3H-alpha chains, but not 3H-labeled tetrameric hemoglobins, were readily degraded; (b) the rate of 3H-alpha chain proteolysis in the cell-free system was at least one-half of that observed for the turnover of newly synthesized alpha chains (t1/2 approximately 6 h) in intact cells; and (c) the ATP-dependent proteolytic activity of both systems was inhibited substantially by certain chemical agents (orthovanadate, N-ethylmaleimide, o-phenanthroline, and phenylmethylsulfonyl fluoride) but only slightly, if at all, by others (epsilon-aminocaproic acid and leupeptin). When excess human erythrocyte ubiquitin was added to the beta-thalassemic cell-free systems, a stimulation in ATP-dependent proteolysis of 3H-alpha chains ranging from 30 to 58% was observed. Conversely, addition of from 1.25 to 2.50 mg/ml affinity-purified rabbit antiubiquitin inhibited almost all (greater than 90%) of the ATP-dependent 3H-alpha chain proteolysis; in control experiments, antiubiquitin neutralized with excess ubiquitin inhibited only 13 to 30% of the total (including ubiquitin-stimulated) ATP-dependent proteolysis.(ABSTRACT TRUNCATED AT 400 WORDS)     

PAN, the proteasome-activating nucleotidase from archaebacteria, is a protein-unfolding molecular chaperone

The proteasome-activating nucleotidase (PAN) from Methanococcus jannaschii is a complex of relative molecular mass 650,000 that is homologous to the ATPases in the eukaryotic 26S proteasome. When mixed with 20S archaeal proteasomes and ATP, PAN stimulates protein degradation. Here we show that PAN reduces aggregation of denatured proteins and enhances their refolding. These processes do not require ATP hydrolysis, although ATP binding enhances the ability of PAN to prevent aggregation. PAN also catalyses the unfolding of the green fluorescent protein with an 11-residue ssrA extension at its carboxy terminus (GFP11). This unfolding requires ATP hydrolysis, and is linked to GFP11 degradation when 20S proteasomes are also present. This unfolding activity seems to be essential for ATP-dependent proteolysis, although PAN may function by itself as a molecular chaperone.     

Energy dependence of autophagic protein degradation in isolated rat hepatocytes

The effect of small changes in intracellular ATP on autophagic flux was studied in isolated rat hepatocytes by using inhibitors of ATP production or by varying the metabolic conditions. The following observations were made. There was a linear relationship between endogenous protein degradation and intracellular ATP, the rate of proteolysis declining with decreasing ATP concentrations. 15% of the maximal proteolysis is either independent of ATP or has a very high affinity for this metabolite. There was a linear relationship between the autophagic sequestration of cytosolic [14C]sucrose and intracellular ATP, the sequestration rate decreasing with decreasing ATP concentrations. ATP depletion did not cause release of [14C]sucrose previously sequestered in autophagosomes and lysosomes at high ATP levels. Intracellular accumulation of chloroquine, used as an indicator of the pH inside lysosomes and other acidic cell compartments, diminished with decreasing cellular ATP content. Amino acids inhibited proteolysis without affecting ATP levels or chloroquine accumulation. We conclude from the high sensitivity of autophagy towards relatively small changes in the concentration of intracellular ATP that, besides amino acids, ATP is a very important factor in controlling the rate of autophagy in rat hepatocytes.     

ATP-stimulated degradation of endogenous proteins in cell-free extracts of BHK 21/C13 fibroblasts. A key role for the proteinase, macropain, in the ubiquitin-dependent degradation of short-lived proteins.

Baby hamster kidney (BHK) 21/C13 cell proteins, labeled with [35S]methionine, [14C]leucine or [3H]leucine in intact cells, were degraded in soluble, cell-free extracts by an ATP-stimulated process. The stimulatory effect of ATP appeared to require ATP hydrolysis and was mediated to a large extent by ubiquitin. Although the cell extracts contained endogenous ubiquitin, supplementation with exogenous ubiquitin increased ATP-dependent proteolysis by up to 2-fold. Furthermore, antibodies against the E1 ubiquitin conjugating enzyme specifically inhibited both conjugation of [125I]ubiquitin to endogenous proteins and ATP/ubiquitin-dependent proteolysis. Addition of purified E1 to antibody-treated extracts restored conjugation and proteolysis. Proteins containing the amino acid analogues canavanine and azatryptophan were also degraded in vitro by an ATP/ubiquitin-dependent process but at a rate up to 2-fold faster than normal proteins. These results indicate that soluble, cell-free extracts of BHK cells can selectively degrade proteins whose rates of degradation are increased in intact cells. Treatment of cell-free extracts with antibodies against the high molecular weight proteinase, macropain, also greatly inhibited the ATP/ubiquitin-dependent degradation of endogenous proteins. Proteolysis was specifically restored when purified macropain L was added to the antibody-treated extracts. Treatment of cell extracts with both anti-macropain and anti-E1 antibodies reduced ATP/ubiquitin-dependent proteolysis to the same extent as treatment with either antibody alone. Furthermore, proteolysis could be restored to the double antibody treated extracts only after addition of both purified E1 and macropain. These results provide strong evidence for an important role for macropain in the ATP/ubiquitin-dependent degradation of endogenous proteins in BHK cell extracts.     

The effects of vanadate and molybdate on cathepsin D; relationship to ATP activation of lysosomal proteolysis

The effects of the phosphate analogues, vanadate and molybdate, on the ATP-activated enzyme, cathepsin D, were investigated. Both were found to inhibit proteolysis but this appeared to be the result of non-specific interactions with the protein substrates which result in precipitation, rather than interactions with the enzyme. Inhibition of proteolysis was induced by the same concentration of inhibitors as that which induced precipitation (measured by turbidity), and was dependent on the concentration of substrate. Precipitation did not occur at neutral pH but was maximal below pH 5. High concentrations of salt (greater than 1M KC1) prevented precipitation of proteins by vanadate and molybdate and under these conditions little inhibition of proteolysis was observed even at high inhibitor concentrations. Nonetheless, ATP was found to activate proteolysis catalyzed directly by lysosomal enzymes at acid pH, while vanadate and molybdate inhibited proteolysis in this system and induced precipitation of substrate. These results indicate that inhibition of proteolysis at acid pH by vanadate (or molybdate) has no relationship to inhibition of proteases and/or ATP dependence of such enzymes. However, direct activation of cathepsin D in lysosomes by ATP remains a viable hypothesis.     

Control of proteolysis by norepinephrine and insulin in brown adipocytes: role of ATP,phosphatidylinositol 3-kinase,and p70 S6K

The objective of this study was to evaluate some of the mechanisms by which norepinephrine (NE) and insulin may influence protein degradation in mouse brown adipocytes differentiated in cultures. The effects of NE and insulin, alone or in combination, on three factors known to influence proteolysis (maintenance of cell ATP and 1-phosphatidylinositol 3-kinase (PI 3-kinase) and p70 ribosomal S6-kinase (p70 S6K) activities) were examined. It was proposed that NE affects proteolysis indirectly by decreasing cell ATP from activation of uncoupling protein-1 (UCP1)-dependent mitochondrial respiration. This was tested by comparing the effects of NE and fatty acids (which directly activate UCP1) on proteolysis in brown adipocytes, as well as in pre-adipocytes and 3T3-L1 adipocytes, which do not express UCP1. An inhibitory effect of insulin on proteolysis is observed in both pre-adipocytes and differentiated cells, whereas NE and exogenously added fatty acids inhibit proteolysis only in brown adipocytes. There is a linear relationship between reductions in cell ATP and proteolysis in response to increasing concentrations of NE or fatty acids. PI 3-kinase activity is required for proteolysis, because two selective inhibitors (wortmannin and LY294002) reduce proteolysis in both pre-adipocytes and differentiated cells. This effect is not additive to that of NE, which suggests they affect the same proteolytic pathway. In contrast to NE, insulin increases PI 3-kinase activity and phosphorylation of p70 S6K. Rapamycin, which prevented insulin-dependent increase in phosphorylation of p70 S6K, increases proteolysis in brown adipocytes and antagonizes the inhibitory effect of insulin on proteolysis, but not the inhibitory effect of NE. Thus, insulin inhibits proteolysis via rapamycin-sensitive activation of p70 S6K, whereas the effect of NE appears largely to be a function of decreasing cell ATP content.     

An adenosinetriphosphate-activated hemolytic system. II. Utilization of adenosinetriphosphate in the reaction

Failure to demonstrate ATP¹ utilization in an ATP-activated hemolytic system had been previously reported. In the present study ATP utilization is shown to be associated with the hemolytic reaction and also with the ATP-dependent inactivation of the hemolytic factor of the system by crude, washed, human red cell stroma. Using crude stroma, relatively large ATP utilization occurs and continues, but at a decreasing rate, after inactivation of the hemolytic factor is complete. With purified stroma there is very slight uptake of ATP by the stroma in the presence of hemolytic factor and Mg⁺⁺. This uptake can only be demonstrated by radioactive ATP. Both C¹⁴- and P³²—labelled nucleotide were used for this purpose. In the presence of an excess of stroma the uptake seems to be dependent on the amount of hemolytic factor used. Evidence is given contraindicating the possibility that this uptake is non-specific.     

Heterologously overexpressed, affinity-purified human meprin alpha is functionally active and cleaves components of the basement membrane in vitro

Meprins are astacin-like metalloproteases of renal and intestinal epithelia and embryonic neuroepithelial cells. The full length cDNA of the human meprin alpha subunit has been overexpressed in baculovirus-infected insect cells yielding the tetrameric proprotein which could be proteolytically activated and affinity-purified to homogeneity. Recombinant meprin alpha hydrolyzes the synthetic substrate N-benzoyl-tyrosyl-p-aminobenzoic acid (PABA-peptide) and cleaves by limited proteolysis the basement membrane constituents laminin 1 and laminin 5. This supports a concept that meprin alpha, when basolaterally secreted by human colon carcinoma epithelial cells, increases the proteolytic capacity for tumor progression in the stroma.     

ATP-dependent association between subunits of Clp protease in pea chloroplasts

The chloroplast ATP-dependent Clp protease (EC 3.4.21.92) is composed of the proteolytic subunit ClpP and the regulatory ATPase, ClpC. Although both subunits are found in the stroma, the interaction between the two is dynamic. When immunoprecipitation with antibodies against ClpC was performed on stroma from dark-adapted pea (Pisum sativum L. cv. Alaska) chloroplasts, ClpC but not ClpP was precipitated. However, when stroma was supplemented with ATP, both ClpC and ClpP were precipitated. Co-immunoprecipitation was even more efficient in the presence of ATP-gamma-S, suggesting that the association between regulatory and proteolytic subunits is dependent on binding of ATP to ClpC, but not its hydrolysis. To further test this association, stroma was fractionated by column chromatography, and the presence of Clp subunits in the different fractions was monitored immunologically. When stroma depleted of ATP was fractionated on an ion-exchange column, ClpP and ClpC migrated separately, whereas in the presence of ATP-gamma-S both subunits co-migrated. Similar results were observed in size-exclusion chromatography. To further characterize the precipitated enzyme, its proteolytic activity was assayed by testing its ability to degrade beta-casein. No degradation was observed in the absence of ATP, and degradation was inhibited in the presence of phenylmethylsulfonyl fluoride, consistent with Clp being an ATP-dependent serine protease. The activity of the isolated enzyme was further tested using chimeric OE33 as a model substrate. This protein was also degraded in an ATP-dependent manner, supporting the suggested role of Clp protease as a major housekeeping protease in the stroma.     

A proteolyzed derivative of Escherichia coli phosphofructokinase is no longer sensitive to allosteric effectors and still shows cooperativity in substrate binding

Limited proteolysis of Escherichia coli phosphofructokinase by subtilisin yields a homogeneous derivative. This proteolyzed protein is composed of four polypeptide chains, with a molecular weight of 32 000 as compared to 37 000 for the original enzyme. Removal on each chain of about 5 kdaltons maintains the enzymatic activity and does not change the apparent affinity for the substrates ATP and fructose 6-phosphate. Limited proteolysis, however, affects the cooperativity of fructose 6-phosphate binding: the Hill coefficient is reduced from almost 4 in the native enzyme to only 2 in its proteolyzed derivative. Also, the proteolyzed protein is no longer sensitive to allosteric effectors, activator, or inhibitor. These changes in regulatory properties upon proteolysis are apparently due to the destruction of the effector binding site. The allosteric effector GDP protects phospho-fructokinase against proteolysis and irreversible thermal inactivation; GDP is, however, inefficient in protecting the proteolyzed protein against thermal denaturation. These results suggest that phosphofructokinase may function as a dimer of dimers, in which homotropic and heterotropic allosteric effects are not mediated by the same sets of quaternary interactions.     

Effects of ATP, vanadate, and molybdate on cathepsin D-catalyzed proteolysis

The effects of ATP, vanadate, and molybdate on cathepsin D-catalyzed hydrolysis of proteins and peptides were examined. Hydrolysis of bovine serum albumin, hemoglobin, parathyroid hormone, and a synthetic octapeptide was activated by ATP. Degradation of the protein substrates all had similar ATP concentration dependence, but the magnitude of the activation varied. Kinetic constants for ATP activation were obtained with a synthetic substrate. ATP increased kcat from 0.4 to 2 s-1 but did not change KM. Kact for ATP was 800 microM. Studies with pepstatin-Sepharose confirm that ATP does not alter the substrate binding site on cathepsin D. Pepsin, a homologous aspartate protease, was not activated by ATP. It was also found that vanadate and molybdate inhibit cathepsin D-catalyzed proteolysis. However, this inhibition was dramatically dependent on substrate concentration and was eliminated at high substrate. Hydrolysis of the synthetic peptide was not inhibited at concentrations of molybdate below 50 microM, and above this concentration the peptide precipitated. Protein substrates were also found to precipitate in the presence of molybdate. The ATP dependence of the enzyme was not altered by molybdate or vanadate. These results suggest that inhibition by vanadate and molybdate is related to interactions with the substrate rather than with cathepsin D. It is concluded that ATP activation of cathepsin D may play a physiological role in regulation of proteolysis in lysosomes, but that vanadate and molybdate inhibition of lysosomal proteolysis does not establish ATP dependence.     

Protein conformational change and nucleotide binding involved in regulation of sigmaF in Bacillus subtilis.

We have studied the ability of three mutant forms of SpoIIAA, containing amino acid substitutions at the site of phosphorylation (serine 58), to interact with SpoIIAB. Native gel analysis revealed that SpoIIAAS58A could form a complex with SpoIIAB in the presence of ADP and more strongly in the presence of ATP. SpoIIAAS58N did not form a complex with SpoIIAB in the presence of ADP but displayed some interaction with SpoIIAB in the presence of ATP. SpoIIAAS58D was unable to form a complex with SpoIIAB in the presence of either ADP or ATP. Corresponding differences were found in the behavior of the three mutant proteins when studied by gel permeation with high-performance liquid chromatography and limited proteolysis. SpoIIAAS58A behaved like the wild-type SpoIIAA, SpoIIAAS58D like SpoIIAA-P, and SpoIIAAS58N in a way that was intermediate between the behaviors of SpoIIAA and SpoIIAA-P. Limited proteolysis was also used to show that on binding of ADP or ATP SpoIIAB undergoes a shift in conformation. The affinity of SpoIIAB for ADP and ATP was determined by limited proteolysis in the presence of a wide range of nucleotide concentrations. The results indicated that SpoIIAB has approximately equal affinity for ADP and for ATP.     

The membrane proteome of stroma thylakoids from Arabidopsis thaliana studied by successive in‐solution and in‐gel digestion

From individual localization and large‐scale proteomic studies, we know that stroma‐exposed thylakoid membranes harbor part of the machinery performing the light‐dependent photosynthetic reactions. The minor components of the stroma thylakoid proteome, regulating and maintaining the photosynthetic machinery, are in the process of being unraveled. In this study, we developed in‐solution and in‐gel proteolytic digestion methods, and used them to identify minor membrane proteins, e.g. transporters, in stroma thylakoids prepared from Arabidopsis thaliana (L.) Heynh Columbia‐0 leaves. In‐solution digestion with chymotrypsin yielded the largest number of peptides, but in combination with methanol extraction resulted in identification of the largest number of membrane proteins. Although less efficient in extracting peptides, in‐gel digestion with trypsin and chymotrypsin led to identification of additional proteins. We identified a total of 58 proteins including 44 membrane proteins. Almost half are known thylakoid proteins with roles in photosynthetic light reactions, proteolysis and import. The other half, including many transporters, are not known as chloroplast proteins, because they have been either curated (manually assigned) to other cellular compartments or not curated at all at the plastid protein databases. Transporters include ATP‐binding cassette (ABC) proteins, transporters for K⁺ and other cations. Other proteins either have a role in processes probably linked to photosynthesis, namely translation, metabolism, stress and signaling or are contaminants. Our results indicate that all these proteins are present in stroma thylakoids; however, individual studies are required to validate their location and putative roles. This study also provides strategies complementary to traditional methods for identification of membrane proteins from other cellular compartments.