Increasing the Efficiency of the Accumulation of Recombinant Proteins in Plant Cells: The Role of Transport Signal Peptides

Abstract

The problem with increasing the yield of recombinant proteins is resolvable using different approaches, including the transport of a target protein to cell compartments with a low protease activity. In the cell, protein targeting involves short-signal peptide sequences recognized by intracellular protein transport systems. The main systems of the protein transport across membranes of the endoplasmic reticulum and endosymbiotic organelles are reviewed here, as are the major types and structure of the signal sequences targeting proteins to the endoplasmic reticulum and its derivatives, to plastids, and to mitochondria. The role of protein targeting to certain cell organelles depending on specific features of recombinant proteins and the effect of this targeting on the protein yield are discussed, in addition to the main directions of the search for signal sequences based on their primary structure. This knowledge makes it possible not only to predict a protein localization in the cell but also to reveal the most efficient sequences with potential biotechnological utility.

Keywords: plant expression system; recombinant protein; transport signal peptide.

Figure 1

The cotranslational protein targeting pathway: (1) The SRP binds a signal sequence (dark blue) as it emerges from a ribosome to form the RNC–SRP complex; (2) the RNC–SRP complex docks with the ER membrane by binding to a cognate SRP receptor (SR, consisting of α and β subunits); (3) subsequently, the RNC is transferred from SR–SRP to translocon Sec61, resulting in the intercalation of the signal sequence into the translocon pore and its opening; (4) the nascent chain is translocated through the pore, and the SRP disengages from the SR; (5) concomitantly with translocation, the signal peptidase (SPase) and oligosaccharyltransferase (OST) enzyme complexes are recruited to the translocon to cleave the SP and add N-linked glycans to the nascent chain, respectively. The termination of the protein synthesis releases the nascent chain from the ribosome; the translocation is completed; and the protein folds in the ER lumen (adapted from [38] with permission).

Figure 2

General structure of an SP. It is composed of three main parts: (1) an N-terminal region (a positively charged domain); (2) H-region (the hydrophobic core forming an α-helix); and (3) a C-terminal region (a cleavage site forming a β-sheet). The initial part of the protein, important to protein secretion, is referred to as the pro-region. Cleavage occurs within the AXA or VXA motif (adapted from [19], with permission).

Figure 3

Mitochondrial and chloroplast protein import machineries. (A) Mitochondrial transmembrane protein transport. TOM, translocon of the outer mitochondrial membrane; TIM, translocase of the inner mitochondrial membrane; PAM, presequence translocon-associated motor; and MPP, mitochondrial processing peptidase. Approximate molecular weights (kDa) are indicated on individual TOM, TIM, and PAM components. See the main text for mechanistic details (adapted from [135], with permission). (B) Core components of TOC–TIC import machinery of chloroplasts. A newly synthesized preprotein is targeted to the TOC complex at the outer membrane by the binding of its transit peptide to receptors Toc34 and Toc159 with GTPase activities. The targeting is aided by the cytosolic chaperone complexes of Hsp70 and Hsp90. The GTPase activity of the receptors is required for the transport of the preprotein through the TOC and TIC transmembrane channels formed by TOC75 and TIC20/21, respectively. The TOC–TIC supercomplex assembled through the binding of Tic236 to Toc75 and of Tic110 to the TIC complex facilitates the direct transport of the preprotein from the cytosol to the chloroplast stroma. Tic110 and Tic40 (40), together with the chloroplast chaperones Hsp70, Hsp93, and Hsp90, form the ATP-dependent protein motor performing import into the chloroplast stroma. After the import, the transit peptide is cleaved by stromal processing peptidase (SPP) (adapted from [159], with permission).

Figure 4

Specific features of the structure of mitochondrial and chloroplast SPs. (A) Eukaryotic signal sequences depending on the target organelle: the ER SP has a common tripartite structure; the mitochondrial-targeting peptide is composed of a matrix signal and intramitochondrial sorting signal; and the chloroplast transit peptide comprises the stroma-targeting peptide and thylakoid-targeting peptide. The intramitochondrial sorting signal and thylakoid-sorting signal share a common tripartite structure. Upward arrows denote the cleavage site, and the horizontal arrow denotes a β-sheet (adapted from [19], with permission). (B) The relation between the chloroplast transit peptide and mitochondrial presequence. The N-terminal specificity domain (NSD) of mitochondrial presequences contains multiple arginine residues (MAR) and a moderately hydrophobic sequence motif (MHSM). The removal of the MAR is sufficient to switch the targeting specificity from mitochondria to chloroplasts. Conversely, the incorporation of both the MAR and MHSM into the NSD of chloroplast transit peptides changes the targeting specificity from chloroplasts to mitochondria. The insertion of the MAR or MHSM alone into a transit peptide results in cytosolic localization or chloroplast targeting, respectively. C-terminal translocation domains (CTDs) are interchangeable between these targeting signals (adapted from [170], with permission).