We present protocols herein for high-yield isolation of physiologically active thylakoids and protein transport assays for the chloroplast twin arginine translocation (cpTat), secretory (cpSec1), and signal recognition particle (cpSRP) pathways.
Chloroplasts are the organelles in green plants responsible for carrying out numerous essential metabolic pathways, most notably photosynthesis. Within the chloroplasts, the thylakoid membrane system houses all the photosynthetic pigments, reaction center complexes, and most of the electron carriers, and is responsible for light-dependent ATP synthesis. Over 90% of chloroplast proteins are encoded in the nucleus, translated in the cytosol, and subsequently imported into the chloroplast. Further protein transport into or across the thylakoid membrane utilizes one of four translocation pathways. Here, we describe a high-yield method for isolation of transport-competent thylakoids from peas (Pisum sativum), along with transport assays through the three energy-dependent cpTat, cpSec1, and cpSRP-mediated pathways. These methods enable experiments relating to thylakoid protein localization, transport energetics, and the mechanisms of protein translocation across biological membranes.
Nearly all of the proteinaceous machinery responsible for proper chloroplast function must be translocated from the cytosol1. At the chloroplast envelopes, protein substrates are imported through the translocon of the outer membrane (TOC) and the translocon of the inner membrane (TIC)2. Further targeting to the thylakoid membrane and lumen occurs through the twin arginine translocation (cpTat)3, the secretory (cpSec1)4, the signal recognition particle (cpSRP)5, and the spontaneous insertion pathways6. A method for the high-yield isolation of physiologically active chloroplasts and thylakoid membranes is necessary to measure the energetics and kinetics of a translocation event, to understand the diverse transport mechanisms in each pathway, and to localize a particular protein substrate of interest to any of the six distinct compartments of the chloroplast.
The isolation of membranes from the chloroplast offers better experimental control over environmental factors (such as salt and substrate concentrations, the presence of ATP/GTP, and pH conditions) that affect the measurement of transport energetics and kinetics. This in vitro environment lends itself to the exploration of mechanistic details of translocation for the same reasons. In addition, while predictive software for localization of chloroplast proteins has improved7,8, in vitro transport assays provide a quicker method for confirmation over microscopy-based fluorescent assays that require a genetically encoded fluorescent tag, plant transformation and/or specific antibodies. Here, we present protocols for chloroplast and thylakoid isolations from peas (Pisum sativum), as well as for transport assays optimized for each of the energy-dependent thylakoid translocation pathways.
1. Initial Materials
2. Chloroplast Isolation and Quantification
Note: The first step in preparation of thylakoids is the isolation of intact chloroplasts10. All materials should be kept cold during preparation. Resuspension of chloroplasts should be handled gently, as breakage at this step can severely limit subsequent thylakoid yield.
3. Isolation of Thylakoids
Note: Thylakoids are prepared by hypotonic lysis of intact chloroplasts. This is achieved by exposing the chloroplasts to a hypotonic buffer lacking sorbitol. Isolated thylakoids can be used for assaying any of the translocation pathways, but stromal extract (SE) must also be isolated during this preparation if either cpSec1 or cpSRP pathways are to be investigated.
4. Stromal Extract Recovery and Concentration
5. Transport through the cpTat Pathway
Note: Unlike the cpSec1 or cpSRP, the cpTat pathway does not require soluble components or exogenously added energy sources; only the light-driven proton motive force is necessary3. Therefore, only isolated thylakoids and substrate protein are required for the assay. Typical substrates are intermediate forms of the 17 kDa (as seen in Figure 1) and 23 kDa subunits of the oxygen evolving complex, iOE17 and iOE23, respectively, but precursor forms, prOE17 and prOE23, can also be successfully transported. Precursor forms have the entire bipartite N-terminal targeting sequence, while intermediate forms have only the thylakoid targeting sequence.
6. Transport through the cpSec1 Pathway
Note: Transport through the cpSec1 translocon requires the stromal protein cpSecA112,13, which can be procured via overexpression in E. coli14,15 or recovered by concentrating stroma during thylakoid isolation. A typical substrate is the 33 kDa subunit of the oxygen evolving complex (prOE33), as seen in Figure 2.
7. Insertion through the cpSRP Pathway
Note: The cpSRP-mediated integration of light harvesting complex proteins (LHCP) seen in Figure 3 requires cpSRP54, cpSRP43, and cpFtsY16. These components are supplied to the transport reaction through concentrated stromal extract, as described for the cpSec1 transport protocol.
To gauge amount of substrate successfully transported, it is useful to include one or more "percent input" lanes. For the data presented below, 10% of the final transport reaction without thylakoids was used. This "percent input" also helps to visualize the size of the precursor substrate. The percentage represents a known, defined amount of substrate with which to compare transported substrate against and can be scaled up or down as necessary using initially prepared protein. Additionally, it is advisable to load less than 4 µg of Chl equivalents in a single lane on 0.75 mm polyacrylamide gels to avoid band warping and smearing. All substrates below were prepared and radiolabeled using in vitro translation kits.
Transport of iOE17 through cpTat pathway
Figure 1. Transport of iOE17through the cpTat pathway. Fluorograph of [3H]-iOE17 transport assay and thermolysin treatment of non-transported substrate. Please click here to view a larger version of this figure.
Successful cpTat transport of most Tat substrates can be detected by a size shift upon cleavage of the N-terminal signal peptide3. In substrates where no cleavable signal peptide exists18,19, protease treatment is required to reveal the substrate that crossed the membrane, thereby becoming inaccessible to digestion by the protease. In Figure 1, transport of iOE17 results in a size shift of approximately 2-3 kDa between the introduced substrate and the mature processed form. Non-transported substrate is degraded through protease treatment (lane 4).
Transport of prOE33 through cpSec1 pathway
Figure 2. Transport of prOE33 through cpSec1 pathway. Autoradiograph of [35S]-prOE33 transport assay using SE, purified cpSecA1, or both simultaneously. Please click here to view a larger version of this figure.
Transport through the cpSec1 pathway (Figure 2) can be evaluated through a size shift in the mature substrate if the substrate contains a cleavable thylakoid targeting signal, as well as protease protection of transported substrate20.
Integration of prLHCP through cpSRP pathway
Figure 3. Integration of prLHCP through cpSRP pathway. Autoradiograph of [35S]-prLHCP and digestion to the product mLHCP-D after insertion into the thylakoid membrane. Please click here to view a larger version of this figure.
Insertion of prLHCP into the thylakoid membrane via the cpSRP pathway can be evaluated through thermolysin digestion. A size shift of approximately 1.5-2 kDa is indicative of successful membrane insertion as the membrane protects the uncleaved mature protein from complete proteolysis15. In Figure 3, the protease-protected product mLHCP-D is clearly visible.
Chloroplast and Thylakoid isolation
Excessive breakage can result in poor chloroplast isolation and thus poor thylakoid yield after separation in the gradient. It is best to homogenize the harvested tissue gently by ensuring that all material is submerged before blending and pulsing in 15 s cycles until fully homogenized. If necessary, use multiple shorter rounds of blending with less tissue in each round.
Refrigerating all materials that come into contact with harvested tissue helps isolated chloroplasts to retain activity up to 2 hours. It is important to keep the chloroplasts on ice in the dark after isolation as well.
It is crucial to include magnesium in buffers contacting isolated thylakoids. Not doing so results in granal destacking and critically affects the generation of proton motive force upon illumination21. Thylakoids have been used in transport reactions up to 2 hours after isolation when kept on ice and in the dark.
Optimizing Transport Reactions
Isolated thylakoids settle rapidly and can result in unequal Chl equivalents between reactions. As such, it is important to mix the thylakoids thoroughly prior to use, especially when setting up a large number of samples.
The Tat Pathway
Transport efficiency through the Tat pathway can be reduced upon excessive washing of the thylakoids since Tha4 (TatA) can be partially extracted from the membrane22. In such cases, it is helpful to reduce thylakoid wash steps prior to the transport reaction. Additionally, longer illumination times can improve detection where substrates are poorly transported under typical conditions.
The cpSec1 Pathway
When assaying the cpSec1 pathway, the amount of cpSecA1 in concentrated SE should support transport, but that transport may be weak. Efficient transport in isolated thylakoids may benefit from the addition of purified cpSecA1 for certain proteins, though stromal chaperones in SE may also help increase the efficiency of transport15. Further, preparations of cpSecA1 protein should not be frozen prior to usage in transport, as this reduces transport activity. Similarly, frozen SE is less efficacious than freshly prepared extract. As in Tat transport, longer incubation periods in the transport mix can help with difficult substrates.
The cpSRP Pathway
While reconstitution of cpSRP transport using individual components has been performed23, it is convenient to supply cpSRP43, cpSRP54, and cpFtsY with SE. Thermolysin resistance is the most stringent criteria for LHCP integration16,17, but alkaline extraction may be performed as well17. While precursors can often be frozen and stored at -80 °C for many transport reactions, prLHCP prepared by in vitro synthesis should be used for transport immediately after synthesis without freezing.
Characterization of a Novel Substrate's Targeting Pathway
In cases where the translocation pathway taken by a novel substrate is unknown, it is advisable to first investigate possible signal peptides in silico using the amino acid sequence of the precursor or intermediate form. The cpTat pathway typically requires a specific twin arginine consensus motif in the signal peptide and no ATP for successful transport under PAR3. Unlike the Tat pathway, the cpSec1 pathway requires ATP and the cpSecA1 protein. Failed transport in conditions lacking these components suggests the cpSec1 pathway20. The cpSRP pathway requires the signal recognition particle found in the SE. Failed transport using purified cpSecA1 and ATP, but a lack of twin arginine consensus motif in the signal peptide, suggests the cpSRP pathway23.
The authors have nothing to disclose.
This manuscript was prepared with funding by the Division of Chemical Sciences, Geosciences, and Biosciences, 408 Office of Basic Energy Sciences of the US Department of Energy through Grant DE-SC0017035
Pisum sativum seeds | Seedway LLC, Hall, NY | 8686 – Little Marvel | |
Miracloth | Calbiochem, Gibbstown, NJ | 475855-1 | |
80% Acetone | Sigma, Saint Louis, MO | 67-64-1 | |
Blender with sharpened blades | Waring Commercial | BB155S | |
Polytron 10-35 | Fischer Sci | 13-874-617 | |
Percoll | Sigma, Saint Louis, MO | GE17-0891-01 | |
Beckman J2-MC with JA 20 rotor | Beckman-Coulter | 8043-30-1180 | |
Sorvall RC-5B with HB-4 rotor | Sorvall | 8327-30-1016 | |
100 mM dithiothreitol (DTT) in 1xIB | Sigma, Saint Louis, MO | 12/3/83 | Can be frozen in aliquots for future use |
200 mM MgATP in 1xIB | Sigma, Saint Louis, MO | 74804-12-9 | Can be frozen in aliquots for future use |
Thermolysin in 1xIB (2mg/mL) | Sigma, Saint Louis, MO | 9073-78-3 | Can be frozen in aliquots for future use |
HEPES | Sigma, Saint Louis, MO | H3375 | |
K-Tricine | Sigma, Saint Louis, MO | T0377 | |
Sorbitol | Sigma, Saint Louis, MO | 50-70-4 | |
Magnesium Chloride | Sigma, Saint Louis, MO | 7791-18-6 | |
Manganese Chloride | Sigma, Saint Louis, MO | 13446-34-9 | |
EDTA | Sigma, Saint Louis, MO | 60-00-4 | |
BSA | Sigma, Saint Louis, MO | 9048-46-8 | |
Tris | Sigma, Saint Louis, MO | 77-86-1 | |
SDS | Sigma, Saint Louis, MO | 151-21-3 | |
Glycerol | Sigma, Saint Louis, MO | 56-81-5 | |
Bromophenol Blue | Sigma, Saint Louis, MO | 115-39-9 | |
B-Mercaptoethanol | Sigma, Saint Louis, MO | 60-24-2 |