Yesterday I gave an overview of the research projects I’ve worked on. So in that spirit, I give you a ‘Throwback Thursday’ Journal Club article from my own publication record:
Roose, J.L. and Pakrasi, H.B. (2004) Evidence that D1 processing is required for manganese binding and extrinsic protein assembly into Photosystem II. Journal of Biological Chemistry. 279: 45417 – 45422.
This was the first paper I published that was all my own work. We were interested in piecing together the necessary steps for Photosystem II assembly with respect to the lumenal side of the complex where the water-splitting reaction occurs. There are many specific steps in assembling the functional PSII complex, but we were interested in three general things: pD1 processing, manganese cluster binding and extrinsic protein association. What is the order of these steps? Are some of these steps absolutely contingent on others for progression along the PSII assembly pathway? Can some of these steps occur only partially?
The D1 protein is the subunit of PSII found at the center of the complex, which binds many of the necessary cofactors for electron transfer. It is first made in a precursor form (called pD1) that must be cleaved by a specific protease (called CtpA). In cyanobacteria, the C-terminal 16 amino acids of pD1 (on the lumenal side of the thylakoid membrane) are removed to yield the mature D1 protein.
The manganese cluster of PSII is the chemical machinery for the water-splitting reaction. It is comprised of four manganese atoms, one calcium ion and one chloride ion and largely held in place by residues of the D1 protein. Notably, the C-terminal residue of the mature D1 protein is responsible for coordinating at least one of the manganese atoms.
The C-terminus of the D1 protein and the manganese cluster are all located on the lumenal side of the complex. Also in this vicinity, three extrinsic proteins (PsbO, PsbU, PsbV) were known to bind to the complex.
At the time I was working on this project some of the lower-resolution PSII crystal structures were coming out and it was clear from those static pictures that the D1 C-terminus, the manganese cluster and the extrinsic proteins were all tangled together and their assembly (and disassembly for the repair cycle) would all be interrelated.
Here’s the breakdown of how we sorted out some of the details:
Observations: The ∆ctpA mutant, which lacks the D1 processing protease CtpA, contains only the pD1 form and this mutant does not accumulate functional PSII complexes. However, we didn’t know exactly what state of assembly they were in with respect to how much of the manganese cluster was assembled and how many of the extrinsic proteins were associated. It had been previously shown that ∆ctpA cells contained about half as much manganese per chlorophyll as control strains, suggesting at most only two of the four manganese atoms could be assembled into PSII complexes when the D1 protein has not been cleaved.
Hypothesis: In the absence of D1 processing (only pD1 is present), PSII complexes will not be able to fully assemble their manganese clusters. Maybe, only one or two of the manganese atoms will be bound to the complex. The extrinsic proteins may or may not be able to stably associate with complexes containing only the pD1 protein.
Experiment: Isolate pD1-containing PSII complexes and analyze them for manganese content and protein composition.* In the cyanobacterium Synechocystis sp. PCC 6803, the ∆ctpA mutation was combined with another mutation called HT3, in which the large PSII membrane protein CP47 contains a histidine-tag. This histidine-tag acts as a specific hook for purifying PSII complexes for subsequent analysis.
Results: While we obtained similar results for manganese content at the level of membranes in ∆ctpAHT3, no manganese atoms were detectable in at the level of PSII complexes in this mutant. We were able to observe some chlorophyll fluorescence changes upon addition of excess manganese, but never any water-splitting activity. Analysis of the protein components of the ∆ctpAHT3 PSII complexes revealed some key differences relative to the control as well. The PsbO, PsbU and PsbV proteins (all lumenal extrinsic proteins**) were absent from ∆ctpAHT3 PSII. These proteins were found to accumulate within the thylakoid lumen, but were not associated with pD1-containing PSII complexes using a partial membrane solubilization procedure. One protein, Psb27 (also a lumenal extrinsic protein)***, was found to be more abundant in the ∆ctpAHT3 PSII complexes relative to the control.
Conclusions: Our data showed that processing of the pD1 protein is required for manganese cluster assembly in PSII. When only pD1 is present, no manganese atoms are stably associated with PSII, although at least one manganese cluster can access its site to cause the changes in the chlorophyll fluorescence transients we observed. None of the extrinsic proteins (PsbO, PsbU and PsbV) can bind to PSII in the presence of pD1, but these proteins exist as stable soluble proteins within the lumenal space of the thylakoid membranes. The pD1-containing PSII complexes contained the core membrane protein components, but no manganese cluster and none of the extrinsic proteins associated with functional PSII complexes. Altogether this means that the pD1 processing event is an early event in PSII assembly that is absolutely required for manganese cluster assembly and stable association of the extrinsic proteins.
Think Ahead: The Psb27 protein is more abundant on ∆ctpAHT3 PSII, which represent partially-assembled PSII complexes, so maybe this protein only transiently associates with PSII assembly intermediates. It may be a factor required for assembly, but not part of the final complex. The structural data on PSII is great, but we need to remember that PSII exists as a dynamic population with complexes constantly cycling through various states of assembly. Future work must be geared toward developing better tools to analyze the diverse forms of this enzyme in order to piece together the details of the assembly and repair cycles.
Here is the link to the full article: http://www.jbc.org/content/279/44/45417.long
* It sounds too elegant and straightforward, right? That does NOT mean it was technically simple. I started this project as a bright-eyed first semester graduate student rotating in the Pakrasi lab. I tackled it with enthusiasm because no one told me how nearly impossible it would be to obtain the ∆ctpAHT3 PSII complexes I needed for analysis. It took me the better part of 3 years (yeah, yeah, rotations, classes and teaching assignments simultaneously) to optimize the procedures I needed. The ∆ctpAHT3 strain (any strain containing only pD1) was an absolute chore to work with- it grew slowly, was light sensitive, didn’t have much PSII. I was constantly tending to tens of liters and tens of liters-worth of cells. I rejoiced to the point of tears when I recovered even ten micrograms of its PSII (typical yields from much less starting volume can be hundreds of micrograms), and No, there is none of that sample left in a freezer anywhere.
** Notably, the PsbQ protein, another lumenal extrinsic protein which had only recently been identified in cyanobacterial PSII preparations, was also missing from the ∆ctpAHT3 PSII complexes. This protein would be the focus of some of my later investigations as well.
*** No one cared much about the small (11 kDa) Psb27 protein before this result. It had been known as a protein component of PSII, but no one knew what it did. It didn’t even get a letter for its name; PSII researchers had just run out of letters in the alphabet and decided to name that one ‘27’. After seeing that gel result, I had hope that Psb27 would be doing something interesting in PSII assembly. This was a hypothesis that would plague me for the rest of my thesis work. As we Southerners like to say, “It stuck in my craw.” Stay tuned for the exciting conclusion (or just read ahead from the links on the My Research page).