Targeting of a foreign protein into the thylakoid lumen of pea chloroplasts

Meadows, Julie W.; Shackleton, Jamie B.; Hulford, Andrew; Robinson, Colin

doi:10.1016/0014-5793(89)80968-8

Cited by 20 publications

(7 citation statements)

References 13 publications

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“…Fig. 5 shows that in the control tests this chimera is imported into chloroplasts and processed to a stromal intermediate form and a lumenal "mature" form, as found in a previous study (29). In the presence of aminopterin, however, translocation across the thylakoid membrane is almost totally blocked, and the stromal form accumulates together with another smaller polypeptide that probably results from degradation of the stromal intermediate form (35).…”

Section: Stabilization Of Dhfr Blocks Translocation By the Sec Pathway-supporting

confidence: 48%

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The Sec-independent Twin-arginine Translocation System Can Transport Both Tightly Folded and Malfolded Proteins across the Thylakoid Membrane

Hynds

Robinson

1998

Journal of Biological Chemistry

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153

124

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A subset of lumen proteins is transported across the thylakoid membrane by a Sec-independent translocase that recognizes a twin-arginine motif in the targeting signal. A related system operates in bacteria, apparently for the export of redox cofactor-containing proteins. In this report we describe a key feature of this system, the ability to transport folded proteins. The thylakoidal system is able to transport dihydrofolate reductase (DHFR) when an appropriate signal is attached, and the transport efficiency is almost undiminished by the binding of folate analogs such as methotrexate that cause the protein to fold very tightly. The system is moreover able to transport DHFR into the lumen with methotrexate bound in the active site, demonstrating that the ⌬pH-driven transport of large, native structures is possible by this pathway. However, correct folding is not a prerequisite for transport. Truncated, malfolded DHFR can be translocated by this system, as can physiological substrates that are severely malfolded by the incorporation of amino acid analogs.

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Section: Stabilization Of Dhfr Blocks Translocation By the Sec Pathway-supporting

confidence: 48%

“…The presence of the 6xHis-tag has no affect on the stability or import competence of the protein (data not shown). The 33K-DHFR construct contained the presequence of wheat 33K, together with 13 residues of mature 33K protein, linked to mouse DHFR; its construction is described (29).…”

Section: Construction Of the Truncated 23k-dhfr Fusion Protein And Anmentioning

confidence: 99%

The Sec-independent Twin-arginine Translocation System Can Transport Both Tightly Folded and Malfolded Proteins across the Thylakoid Membrane

Hynds

Robinson

1998

Journal of Biological Chemistry

Self Cite

153

124

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“…Indeed, the possibility cannot be ruled out that even parts of the mature protein which are not assumed to actively participate in the actual translocation process, like the hydrophilic domains of thylakoid lumen proteins, might provide a target for internal regulatory mechanisms of the organelle. In order to overcome such potential problems, several passenger proteins of non-plastid origin including blactamase and dihydrofolate reductase (DHFR) have previously been tested in in vitro import assays (Ko and Cashmore 1989;Meadows et al 1989;Hageman et al 1990;de Boer et al 1991;Hynds et al 1998). While both proteins are suitable passengers for import into the chloroplast stroma, only DHFR is translocated across the thylakoid membrane into the lumenal space (Ko and Cashmore 1989;Meadows et al 1989;Hynds et al 1998).…”

Section: Introductionmentioning

confidence: 99%

“…In order to overcome such potential problems, several passenger proteins of non-plastid origin including blactamase and dihydrofolate reductase (DHFR) have previously been tested in in vitro import assays (Ko and Cashmore 1989;Meadows et al 1989;Hageman et al 1990;de Boer et al 1991;Hynds et al 1998). While both proteins are suitable passengers for import into the chloroplast stroma, only DHFR is translocated across the thylakoid membrane into the lumenal space (Ko and Cashmore 1989;Meadows et al 1989;Hynds et al 1998). These results have led us to search for a further non-chloroplast protein which, besides being a suitable passenger for thylakoid transport experiments in vitro, might also allow in vivo import studies.…”

Section: Introductionmentioning

confidence: 99%

Targeting of EGFP chimeras within chloroplasts

2003

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We have tested the potential of EGFP, a derivative of the green fluorescent protein (GFP), as a passenger protein for the analysis of protein transport processes across the thylakoid membranes in chloroplasts. In contrast to the majority of fusion proteins commonly used in such studies, EGFP is not of plant origin and can therefore be assumed to behave like a "neutral" passenger protein that is unaffected by any internal plant regulatory circuits. Our in vitro transport experiments clearly demonstrate that EGFP is a suitable passenger protein that can be correctly targeted either to the stroma or to the thylakoid lumen if fused to the appropriate transit peptide. The transport of EGFP across the thylakoid membrane shows, however, a clear pathway preference. While the protein is efficiently targeted by the deltapH/TAT pathway, transport by the Sec pathway is barely detectable, either with isolated thylakoids or with intact chloroplasts. This pathway specificity suggests that EGFP is folded immediately after import into the chloroplast stroma, thus preventing further translocation across the thylakoid membrane by the Sec translocase. The data obtained provide a good basis for the development of molecular tools for transport studies using EGFP as a passenger protein. Furthermore, plant lines expressing corresponding EGFP chimeras are expected to allow in vivo studies on the transport and sorting mechanisms involved in the biogenesis of the chloroplast.

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“…The presence of sufficient information in the thylakoid transfer domain for routeing of passenger proteins was suggested by the proper routeing in vitro of the 33 kDa subunit-DHFR fusion protein (Ko and Cashmore, 1989;Meadows et al, 1989). The absence of proper routeing in vitro of all the other fusion proteins, however, made this conclusion questionable.…”

Section: Discussionmentioning

confidence: 99%

Protein targeting towards the thylakoid lumen of chloroplasts: proper localization of fusion proteins is only observed in vivo.

et al. 1991

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Routeing of fusion proteins to the thylakoid lumen of the chloroplast was compared in vitro and in vivo. The Escherichia coli protein beta‐lactamase was used as a passenger to study this intraorganellar sorting process. The first step, translocation of beta‐lactamase into the chloroplast stroma, occurs properly both in vitro and in vivo and is dependent on the presence of a transit peptide in the protein construct. The second step, targeting towards the thylakoid lumen, is more complicated as was also observed previously when other passenger proteins were used. In vitro, the presence of a thylakoid transfer domain is not enough for routeing and proper processing. Only when the complete thylakoid lumen precursor plastocyanin was fused to beta‐lactamase was the fusion protein processed adequately, but routeing was still incomplete. However, in vivo, the information present in the thylakoid transfer domain was the only requirement for proper transport towards the thylakoid lumen. These data show that in vivo, the only requirement for targeting of passenger proteins towards the thylakoid lumen is the presence of a transit peptide and a thylakoid transfer domain. Furthermore, we demonstrate that the in vitro import system does not necessarily reflect the in vivo situation with respect to intraorganellar sorting.

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Targeting of a foreign protein into the thylakoid lumen of pea chloroplasts

Cited by 20 publications

References 13 publications

The Sec-independent Twin-arginine Translocation System Can Transport Both Tightly Folded and Malfolded Proteins across the Thylakoid Membrane

The Sec-independent Twin-arginine Translocation System Can Transport Both Tightly Folded and Malfolded Proteins across the Thylakoid Membrane

Targeting of EGFP chimeras within chloroplasts

Protein targeting towards the thylakoid lumen of chloroplasts: proper localization of fusion proteins is only observed in vivo.

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