Peptide transit

As described earlier, translation of the EPSPS mRNA of plants results in the formation of a protein which has an AJ-terminal extension. The AJ-terminal extension, referred to as the chloroplast transit peptide, is necessary and sufficient for the import of the preprotein by the chloroplast. Once imported by the chloroplast, the transit peptide is cleaved releasing the mature enzyme. As expected, introduction of the EPSPS transit peptide to other protein sequences results in the importation of the fusion protein by the chloroplast. 

Van den Broek, G., Timko, M.P., Kausch, A.P., Cashmore, A.R., Van Montagu, M. Herrera-Estrella, L. (1985). Targeting of a foreign protein to chloroplasts by fusion to the transit peptide from the small subunit of ribulose 1,5-biphosphate carboxylase. Nature, 313, 358-63. 

Although carotenogenesis in plants takes place in plastids, all of the carotenoid biosynthesis genes are nuclear encoded and their polypeptide products are imported into the plastids. Therefore, they contain a N-terminal transit peptide sequence. For example, the size of the transit peptide of PSY from ripe tomato fruit is approximately 9 kDa, corresponding to about 80 amino acid residues (Misawa et al, 1994). 

Human hemoglobin (< + P) CaMVenhanced 35S promoter/ CaMV 35S terminator Transit peptide of small subunit of RubisCO of Pisum sativum N. tabacum 0.05% of seed protein 37... 

Chloro pink, 9 310-311 Chloroplast transit peptide, 72 489 TV-Chloropolyacrylamides, 7 316 Chloroprene, 6 242, 246. See also 2-Chloro-1,3- butadiene from butadiene, 4 369 chlorocarbon/chlorohydrocarbon of industrial importance, 6 227t copolymerization of, 79 829-830 end use of chlorine, 6 134t removal in vinyl chloride manufacture, 25 642... 

Plants contain phytoferritins, which accumulate in non-green plastids1 in conditions of iron loading. They are targeted to the plastids by a putative-transit peptide at their N-terminal extremity, and possess the specific residues for ferroxidase activity and iron nucleation, found in mammalian H-type or L-type ferritin subunits. 

SIGNAL from sig peptide TRANSIT from transit peptide CHAIN from mat peptide... 

Although the chloroplast transit peptide has been studied relatively thoroughly, further intrachloroplastic sorting signals have not been well understood. Some of them are introduced below in the context of describing the known sorting pathways (Keegstra and Cline, 1999 Chen and Schnell, 1999 Cline and Henry, 1996 Kouranov and Schnell, 1996). 

However, there is another class of outer membrane proteins (such as Toc34) that do not have a cleavable transit peptide. These proteins appear to be relatively small. A study suggested that their signal resides on the N-terminal 30 residues (Li and Chen, 1996). The signal consists of a positively charged N-terminal portion followed by a hydrophobic core, although it is not certain whether this feature is general. 

The inner envelope membrane proteins have a cleavable N-terminal transit peptide, as well as some hydrophobic domain (s) in their mature portion. There are two possibilities on the role of this hydrophobic domain it may work as an N-terminal signal peptide after the translocation into the stroma and the subsequent cleavage of the transit peptide. Alternatively, it may work as a stop-transfer signal. One more important question is how the distinction is made between the outer membrane proteins, the inner membrane proteins, and the thylakoid membrane proteins. It is still an enigma. 

All thylakoidal proteins seem to be first translocated into the stroma through the previously mentioned general import pathway all of them have a cleavable N-terminal transit peptide. However, there are at least four different pathways into the thylakoid membrane (Robinson et al, 1998 Schnell, 1998). Most of them are reminiscent of the pathways of bacteria, described in Section II,B,1. It is not surprising because chloroplasts are most likely evolved from a prokaryotic endosymbioint, but there are certain differences. 

Emanuelsson, O., Nielsen, H., and von Heijne, G. (1999). ChloroP, a neural network-based method for predicting chloroplast transit peptides and their cleavage sites. Protein Sd. 8, 978—984. 

A transit peptide consisting of a hydrophobic 66 amino acid long peptide interspersed with positively charged residues has been identified and sequenced [52]. This is initially attached to the 99 amino acids of the mature plastocyanin, and is responsible for taking the plastocyanin across membranes into the thylakoid region of the chloroplast. 

The coding sequences of a- and P-globins of human hemoglobin have been fused to the sequence of the chloroplastic transit peptide of the small subunit of Rubisco. These proteins were then coexpressed in transgenic tobacco plants, resulting in the production of a functional form of tetra-meric hemoglobin. The results demonstrate that a complex multimeric protein such as recombinant human hemoglobin can be obtained from tobacco in a functional form. 

Import into the endoplasmic reticulum requires an N-terminal signal sequence that contains a long stretch of hydrophobic amino acids. The mitochondrial transit peptide is a hydrophilic sequence rich in serine and threonine, with regularly spaced basic amino acids. Import into the ER requires the signal recognition particle and its receptor, but mitochondrial import does not require the SRP and presumably uses a different receptor. Import into mitochondria requires a membrane potential, but import into the ER does not. 

Y Gavel, GA von Hejne. A conserved cleavage-site motif in chloroplast transit peptides. FEBS Lett 261 455-458, 1990. 

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