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A monomeric, tightly folded stromal intermediate on the ΔpH-dependent thylakoidal protein transport pathway

A monomeric, tightly folded stromal intermediate on the ΔpH-dependent thylakoidal protein transport pathway
A monomeric, tightly folded stromal intermediate on the ΔpH-dependent thylakoidal protein transport pathway

Two distinct mechanisms have been previously identified for the transport of proteins across the chloroplast thylakoid membrane, one of which is unusual in that neither soluble factors nor ATP are required; the system requires only the transthylakoidal ΔpH. We have examined this mechanism by studying the properties of one of its substrates: the extrinsic 23-kDa protein (23K) of photosystem II. Previous work has shown that this protein can be transported into isolated thylakoids as the full-length precursor protein; we show that the stromal import intermediate form of this protein is similarly translocation-competent. Gel filtration tests indicate that the stromal intermediate is probably monomeric. Protease sensitivity tests on both the initial in vitro translation product and the stromal import intermediate show that the presequence is highly susceptible to digestion whereas the mature protein is resistant to high concentrations of trypsin. The mature protein becomes very sensitive to digestion if unfolded in urea, or after heating, and we therefore propose that the natural substrate for this translocation system consists of a relatively unfolded presequence together with a tightly folded passenger protein. The ability of thylakoids to import pre-23K is destroyed by prior treatment of the thylakoids with low concentrations of trypsin, demonstrating the involvement of surface-exposed proteins in the import process. However, we can find no evidence for the binding of pre-23K or i23K to the thylakoid surface, and we therefore propose that the initial interaction of these substrates with the thylakoidal translocase is weak, reversible, and probably ΔpH-independent. In the second phase of the translocation mechanism, the ΔpH drives either the translocation and unfolding of proteins, or the translocation of a fully folded protein.

0021-9258
1663-1669
Creighton, Alison M.
a0891d46-87ed-4cc3-998f-2811e1651f5e
Hulford, Andrew
540b2f40-a8d7-45c3-a466-1a5f99a13f2d
Mant, Alexandra
63319e45-deeb-45ad-a30d-e05b42052a0d
Robinson, David
16719d43-cde4-44c2-8cfa-83422af342cf
Robinson, Colin
678e0157-d628-44e8-83de-3591b07c673f
Creighton, Alison M.
a0891d46-87ed-4cc3-998f-2811e1651f5e
Hulford, Andrew
540b2f40-a8d7-45c3-a466-1a5f99a13f2d
Mant, Alexandra
63319e45-deeb-45ad-a30d-e05b42052a0d
Robinson, David
16719d43-cde4-44c2-8cfa-83422af342cf
Robinson, Colin
678e0157-d628-44e8-83de-3591b07c673f

Creighton, Alison M., Hulford, Andrew, Mant, Alexandra, Robinson, David and Robinson, Colin (1995) A monomeric, tightly folded stromal intermediate on the ΔpH-dependent thylakoidal protein transport pathway. The Journal of Biological Chemistry, 270 (4), 1663-1669. (doi:10.1074/jbc.270.4.1663).

Record type: Article

Abstract

Two distinct mechanisms have been previously identified for the transport of proteins across the chloroplast thylakoid membrane, one of which is unusual in that neither soluble factors nor ATP are required; the system requires only the transthylakoidal ΔpH. We have examined this mechanism by studying the properties of one of its substrates: the extrinsic 23-kDa protein (23K) of photosystem II. Previous work has shown that this protein can be transported into isolated thylakoids as the full-length precursor protein; we show that the stromal import intermediate form of this protein is similarly translocation-competent. Gel filtration tests indicate that the stromal intermediate is probably monomeric. Protease sensitivity tests on both the initial in vitro translation product and the stromal import intermediate show that the presequence is highly susceptible to digestion whereas the mature protein is resistant to high concentrations of trypsin. The mature protein becomes very sensitive to digestion if unfolded in urea, or after heating, and we therefore propose that the natural substrate for this translocation system consists of a relatively unfolded presequence together with a tightly folded passenger protein. The ability of thylakoids to import pre-23K is destroyed by prior treatment of the thylakoids with low concentrations of trypsin, demonstrating the involvement of surface-exposed proteins in the import process. However, we can find no evidence for the binding of pre-23K or i23K to the thylakoid surface, and we therefore propose that the initial interaction of these substrates with the thylakoidal translocase is weak, reversible, and probably ΔpH-independent. In the second phase of the translocation mechanism, the ΔpH drives either the translocation and unfolding of proteins, or the translocation of a fully folded protein.

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Published date: 1995

Identifiers

Local EPrints ID: 413062
URI: https://eprints.soton.ac.uk/id/eprint/413062
ISSN: 0021-9258
PURE UUID: 093d8e75-f4b6-42df-98db-32c1cc124bf1

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Date deposited: 14 Aug 2017 16:31
Last modified: 13 Mar 2019 20:14

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Contributors

Author: Alison M. Creighton
Author: Andrew Hulford
Author: Alexandra Mant
Author: David Robinson
Author: Colin Robinson

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