Protein Transport Pathways in A.thaliana Thylakoid
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Protein Transport Pathways in A.thaliana Thylakoid

Chloroplasts are exceptionally complex organelles found ubiquitously in Plant and Algal cells. Chloroplasts contain at least six suborganellar compartments: Outer and Inner membranes, Intermembrane Space, Stroma, and Thylakoid membrane and Lumen, all of which require specific trafficking systems. The Thylakoid membrane of the Chloroplast accounts for the bulk of the Chloroplast lipid content and contains the abundant proteins associated with light capture and photosynthetic electron transport. The targeting systems of the Thylakoid appear to be conserved from the Protein export systems that exist in Gram-negative bacteria. The Thylakoid membrane contains four especially prominent complexes (Photosystems I and II, Cytochrome b/f complex and the ATP Synthase), each of which contains a mixture of Chloroplast-encoded and Nucleus-encoded subunits. The import and sorting of proteins to this membrane and the lumenal space takes place by at least four independent pathways which are designated as Sec-dependent, SRP (Signal Recognition Particle)-dependent, DpH/Tat (Twin-Arginine Translocation) -dependent, or ‘Spontaneous’.  Each of them operates with a unique mechanism and is specific for a distinct subset of Thylakoid proteins. Thylakoid lumen proteins are almost invariably encoded by the nucleus, necessitating complete transport across both of the Chloroplast membrane systems. There are two sequential, independent translocation events and lumenal proteins are synthesized with bipartite presequences that contain two distinct targeting signals in tandem. The first ‘transit’ peptide appears to be structurally and functionally equivalent to those of imported Stromal proteins, and it seems that lumenal proteins are similarly imported into the stroma by the default as TOC (Translocon at the Outer envelope membrane of Chloroplasts)/TIC (Translocon at the Inner envelope membrane of Chloroplasts) pathway. The two signal peptides are removed by two different processing peptidases, one resident in the Stroma (SPP (Stromal Processing Peptidase)) and the other in the Thylakoid lumen (TPP (Thylakoidal Processing Peptidase)) (Ref. 1 & 2).

Although all four Thylakoid transport pathways were shown to accept membrane proteins as substrates, two of them, notably the ‘Spontaneous’ and the SRP-dependent transport, are exclusively targeting integral membrane proteins and are thus specific for the insertion of proteins into the Thylakoid membrane. SRP-dependent transport in Chloroplasts presumably provides the major pathway for the integration of polytopic Thylakoid membrane proteins, in analogy to its role in the bacterial system. The main representative of this class of Thylakoid proteins is LHCP, the apoprotein of the light-harvesting complex associated with Photosystem II, and this protein is the model substrate for the analysis of SRP-dependent protein transport in chloroplasts. It carries a transit peptide which has solely Stroma targeting function and is thus completely removed by a SPP. Subsequent targeting of LHCP to the Thylakoids and insertion into the membrane is mediated by internal, uncleaved signals in the mature body of the protein. Thylakoid targeting of LHCP depends on three stromal factors, cpSRP54 which shows sequence homology to the 54 kDa subunit of the cytosolic signal recognition particle and its bacterial counterpart Ffh, cpSRP43 for which no homolog is known from other systems, and cpFtsY, the chloroplast homolog to the bacterial SRP receptor protein FtsY. In addition, Alb3, a Thylakoid membrane protein with homology to bacterial YidC and mitochondrial Oxa1p, is required. Protein transport by the SRP-dependent pathway is energized by hydrolysis of GTP and by the trans-Thylakoidal proton gradient in an as yet unknown manner. After import into the chloroplast, the substrate interacts with SRP54 and SRP43 and their partner protein, FtsY. These factors direct LHCP to the membrane and insertion occurs in a process that depends on membrane-bound Alb3.The available evidence suggests that SRP of chloroplasts lacks any structural RNA, in contrast to its counterparts in the eukaryotic and bacterial cytosols (Ref. 3, 4 & 5).

Direct or ‘Spontaneous’ protein insertion into the Thylakoid membrane is restricted to a specific class of membrane proteins with strikingly similar structure and membrane topology. Proteins belonging to this group are all Integral membrane proteins, and their main feature is that they do not require any known protein transport machinery, nor energy, for insertion into Thylakoid membranes. This is an unusual pathway, which is unique to chloroplasts, and the absence of any known energy requirement or essential targeting factor suggests the possibility of a spontaneous insertion mechanism. Examples of proteins that insert ‘Spontaneously’ are the W and X subunits of Photosystem II (PsbW and PsbX), PsaK from Photosystem I and the SecE subunit (cpSecE). It was first decribed for CFo-II, the only nuclear-encoded component of the CFo-membrane assembly of chloroplast ATP synthase (Ref. 6).

The other two protein transport pathways operating at the Thylakoid membrane, notably the Sec and DpH/Tat-dependent pathways are responsible for the translocation of hydrophilic proteins into the Thylakoid lumen, although for both pathways also the transport of a few integral membrane proteins has been described. Sec-dependent protein transport across the Thylakoid membrane is largely similar to the main secretory pathway in bacteria, i.e. it depends on nucleoside triphosphates and a stromal homolog to the bacterial SecA protein. It does not require the proton gradient across the Thylakoid membrane, although such a membrane potential has a stimulatory effect in some instances. In addition to SecA, two further components of the translocation machinery have been identified, notably the integral Thylakoid proteins SecY and SecE which both show significant homology to their bacterial counterparts and presumably form the translocation pore in the Thylakoid membrane.  Typical substrates of the Thylakoidal Sec-dependent pathway are Plastocyanin, the Photosystem I subunit PsaF, and the 33 kDa protein of the oxygen evolving system, OE33. Sec-mediated translocation can occur only when substrates are in an unfolded state (Ref. 7 & 8).

The alternative means of transport across the thylakoid membrane is provided by the Tat pathway. Substrates for this pathway also bear cleavable amino-terminal signal peptides but these targeting signals are recognized by a translocase that has only recently been characterized in any detail. The system derives its name from a Twin-Arginine motif, located in the amino-terminal region of the signal peptide, which is essential for translocation by this pathway. Curiously, these signal peptides are otherwise very similar to Sec-type signal peptides: they contain clearly identifiable N-, H- and C-domains and similarly end with the Ala-Xaa-Ala consensus motif that specifies cleavage by TPP. The Tat pathway is characterized by a number of unique features. It operates independently of both, soluble factors and nucleoside triphosphates, and is exclusively energized by the proton gradient generated across the Thylakoid membrane upon photosynthetic electron flow. The latter was the reason for its initial designation DpH-dependent pathway. However, the most prominent feature of the Tat pathway is its property to translocate fully folded proteins or protein domains across the Thylakoid membrane which obviously has significant implications on the transport mechanism as well as the structure and function of the translocase. Substrates on the Tat pathway, for example PsbP or PsbQ, have been shown to refold in the stroma and are not believed to interact with any dedicated Tat components until they reach the membrane. Here, they are transported by a membrane-bound Tat system. Three components of the Thylakoidal DpH/Tat transport machinery have been identified to date, called TatA (or Tha4), TatB (or Hcf106), and TatC (or cpTatC). Tha4 and Hcf106 are single-span proteins that share significant homology but nevertheless carry out distinct functions. TatC proteins are believed to have six transmembrane spans. Tat substrates bind preferentially to the Hcf106 and cpTatC subunits, which suggest that these form the initial binding site for substrates. The Tat system is able to translocate substrates in a fully folded state and the Thylakoidal pH is essential for translocation, at least in isolated Thylakoids and intact isolated chloroplasts. Although recent, unexpected developments in the mitochondrial protein import field urge caution in this regard, it seems likely that most components of the chloroplast protein import apparatus have now been identified. Thus, attention has turned to the assignment of specific functions to individual components, and to the elucidation of mechanistic details of the import process. An interesting recent development has been the identification of envelope translocation pathways that exhibit a degree of substrate specificity. In the future, it will be important to define these substrate specificities in greater detail, and to elucidate the properties within the respective transit peptides that form the basis for discrimination (Ref. 9, 10 & 11).