Non-native, N-terminal Hsp70 Molecular Motor Recognition Elements in Transit Peptides Support Plastid Protein Translocation

Chotewutmontri, Prakitchai; Bruce, Barry D.

doi:10.1074/jbc.m114.633586

Cited by 33 publications

(40 citation statements)

References 94 publications

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“…Another possibility is that a translocon component interacts with the main characteristics of amino acids in a certain region of TPs, but does not bind to the motifs with a specific sequence, as in the case of the signal recognition particle that binds to the hydrophobic leader sequences. For example, cpHsc70 interacts with the N-terminal motif of TPs, which has a moderate hydrophobicity conferred by a few hydrophobic amino acids (Lee et al, 2008;Chotewutmontri et al, 2012;Chotewutmontri and Bruce, 2015). A recent study by Teng et al (2012) also supports this notion; they showed that preproteins with two consecutive basic residues are actively imported into older chloroplasts, whereas those with two consecutive acidic residues are not.…”

Section: Discussionsupporting

confidence: 57%

“…Previous studies have shown that the moderate hydrophobicity of the N-terminal region in TPs is important for an early cytosolic step, as well as for recognition by cpHsc70 in the stroma (Lee et al, 2006(Lee et al, , 2008Chotewutmontri et al, 2012;Chotewutmontri and Bruce, 2015). In RbcS-nt, residues ML (amino acids 5-6) and MV (amino acids 11-12) contribute to the moderate hydrophobicity of the N-terminal region.…”

Section: New Sequence Contexts Lead To Changes In Both Composition Anmentioning

confidence: 99%

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Sequence Motifs in Transit Peptides Act as Independent Functional Units and Can Be Transferred to New Sequence Contexts

et al. 2015

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A large number of nuclear-encoded proteins are imported into chloroplasts after they are translated in the cytosol. Import is mediated by transit peptides (TPs) at the N termini of these proteins. TPs contain many small motifs, each of which is critical for a specific step in the process of chloroplast protein import; however, it remains unknown how these motifs are organized to give rise to TPs with diverse sequences. In this study, we generated various hybrid TPs by swapping domains between Rubisco small subunit (RbcS) and chlorophyll a/b-binding protein, which have highly divergent sequences, and examined the abilities of the resultant TPs to deliver proteins into chloroplasts. Subsequently, we compared the functionality of sequence motifs in the hybrid TPs with those of wild-type TPs. The sequence motifs in the hybrid TPs exhibited three different modes of functionality, depending on their domain composition, as follows: active in both wild-type and hybrid TPs, active in wild-type TPs but inactive in hybrid TPs, and inactive in wild-type TPs but active in hybrid TPs. Moreover, synthetic TPs, in which only three critical motifs from RbcS or chlorophyll a/bbinding protein TPs were incorporated into an unrelated sequence, were able to deliver clients to chloroplasts with a comparable efficiency to RbcS TP. Based on these results, we propose that diverse sequence motifs in TPs are independent functional units that interact with specific translocon components at various steps during protein import and can be transferred to new sequence contexts.

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Section: Discussionsupporting

confidence: 57%

Section: New Sequence Contexts Lead To Changes In Both Composition Anmentioning

confidence: 99%

Sequence Motifs in Transit Peptides Act as Independent Functional Units and Can Be Transferred to New Sequence Contexts

et al. 2015

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“…It is also known that stromal Hsp70 acts as a molecular motor facilitating uptake of chloroplast preproteins into the chloroplast stroma (59). However, little nucleotide sequence identity exists between this nuclear encoded protein and Hsc70-2 (ca.…”

Section: Hsp70 Facilitates Targeting Of Cnv Cp To Chloroplastsmentioning

confidence: 99%

“…During or after translocation, the transit peptide is cleaved off by the stromal processing peptidase in the stroma (58) and the mature proteins are then released and folded (53). A stromal HSP70 has also been shown to participate in uptake of preproteins into the chloroplast (59). Previous work in our laboratory has shown that approximately 1 to 5% of the total CNV CP present in a cell during infection of Nicotiana benthamiana is found within chloroplasts (60).…”

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confidence: 98%

Cucumber Necrosis Virus Recruits Cellular Heat Shock Protein 70 Homologs at Several Stages of Infection

Alam

Rochon

2016

J Virol

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RNA viruses often depend on host factors for multiplication inside cells due to the constraints of their small genome size and limited coding capacity. One such factor that has been exploited by several plant and animal viruses is heat shock protein 70 (HSP70) family homologs which have been shown to play roles for different viruses in viral RNA replication, viral assembly, disassembly, and cell-to-cell movement. Using next generation sequence analysis, we reveal that several isoforms of Hsp70 and Hsc70 transcripts are induced to very high levels during cucumber necrosis virus (CNV) infection of Nicotiana benthamiana and that HSP70 proteins are also induced by at least 10-fold. We show that HSP70 family protein homologs are co-opted by CNV at several stages of infection. We have found that overexpression of Hsp70 or Hsc70 leads to enhanced CNV genomic RNA, coat protein (CP), and virion accumulation, whereas downregulation leads to a corresponding decrease. Hsc70-2 was found to increase solubility of CNV CP in vitro and to increase accumulation of CNV CP independently of viral RNA replication during coagroinfiltration in N. benthamiana. In addition, virus particle assembly into virus-like particles in CP agroinfiltrated plants was increased in the presence of Hsc70-2. HSP70 was found to increase the targeting of CNV CP to chloroplasts during infection, reinforcing the role of HSP70 in chloroplast targeting of host proteins. Hence, our findings have led to the discovery of a highly induced host factor that has been co-opted to play multiple roles during several stages of the CNV infection cycle. IMPORTANCEBecause of the small size of its RNA genome, CNV is dependent on interaction with host cellular components to successfully complete its multiplication cycle. We have found that CNV induces HSP70 family homologs to a high level during infection, possibly as a result of the host response to the high levels of CNV proteins that accumulate during infection. Moreover, we have found that CNV co-opts HSP70 family homologs to facilitate several aspects of the infection process such as viral RNA, coat protein and virus accumulation. Chloroplast targeting of the CNV CP is also facilitated, which may aid in CNV suppression of host defense responses. Several viruses have been shown to induce HSP70 during infection and others to utilize HSP70 for specific aspects of infection such as replication, assembly, and disassembly. We speculate that HSP70 may play multiple roles in the infection processes of many viruses. C ucumber necrosis virus (CNV) is a positive-strand RNA virus in the genus Tombusvirus, family Tombusviridae (1). The CNV genome is monopartite and consists of ϳ4.7 kb of positive polarity single-stranded RNA. The genome contains five open reading frames (ORFs) which encode five different proteins: the auxiliary replicase factor (p33), the RNA-dependent RNA polymerase (RdRp) (p92), the coat protein (CP; p41), the movement protein (p21), and the silencing suppressor (p20) (Fig. 1A). The UAG stop codon of the p...

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“…We have shown here that Hsp93 directly binds to transit peptides during preprotein import. Since it has also been suggested that Hsp70 can bind to the N terminus of transit peptides (Chotewutmontri et al, 2012;Chotewutmontri and Bruce, 2015), cpHsc70 may also contribute to transit peptide binding. These two ATPases may prefer different residues/motifs in the transit peptide or provide different modes of translocation force, and, therefore, the double mutant would exhibit additive import defects.…”

Section: Discussionmentioning

confidence: 99%

Chloroplast Hsp93 Directly Binds to Transit Peptides at an Early Stage of the Preprotein Import Process

Huang

Chan

et al. 2015

Plant Physiol.

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Three stromal chaperone ATPases, cpHsc70, Hsp90C, and Hsp93, are present in the chloroplast translocon, but none has been shown to directly bind preproteins in vivo during import, so it remains unclear whether any function as a preproteintranslocating motor and whether they have different functions during the import process. Here, using protein crosslinking followed by ionic detergent solubilization, we show that Hsp93 directly binds to the transit peptides of various preproteins undergoing active import into chloroplasts. Hsp93 also binds to the mature region of a preprotein. A time course study of import, followed by coimmunoprecipitation experiments, confirmed that Hsp93 is present in the same complexes as preproteins at an early stage when preproteins are being processed to the mature size. In contrast, cpHsc70 is present in the same complexes as preproteins at both the early stage and a later stage after the transit peptide has been removed, suggesting that cpHsc70, but not Hsp93, is important in translocating processed mature proteins across the envelope.Most chloroplast proteins are encoded by the nuclear genome as higher M r preproteins that are fully synthesized in the cytosol before being imported into the chloroplast. The import process is initiated by binding of the N-terminal transit peptide of the preprotein to the translocon at the outer envelope membrane of chloroplasts (TOC) complex, in which Toc159 and Toc34 function as receptors and Toc75 is the outer membrane channel. This step is followed by binding of the transit peptide to the translocon at the inner envelope membrane of chloroplasts (TIC) machinery, the central components of which include the Tic20/Tic56/ Tic100/Tic214 channel complex and Tic110. Tic110 functions as the stromal receptor for transit peptides and also as a scaffold for tethering other translocon components (for reviews, see Li and Chiu, 2010;Shi and Theg, 2013;Paila et al., 2015). The actual translocation of the bound preproteins across the envelope is powered by hydrolysis of ATP in the stroma (Pain and Blobel, 1987;Theg et al., 1989), and it is therefore assumed that some stromal ATPase motor proteins bind the preproteins as they emerge from the inner membrane and use the energy of ATP hydrolysis to translocate the preproteins across the envelope into the stroma.

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Non-native, N-terminal Hsp70 Molecular Motor Recognition Elements in Transit Peptides Support Plastid Protein Translocation

Cited by 33 publications

References 94 publications

Sequence Motifs in Transit Peptides Act as Independent Functional Units and Can Be Transferred to New Sequence Contexts

Sequence Motifs in Transit Peptides Act as Independent Functional Units and Can Be Transferred to New Sequence Contexts

Cucumber Necrosis Virus Recruits Cellular Heat Shock Protein 70 Homologs at Several Stages of Infection

Chloroplast Hsp93 Directly Binds to Transit Peptides at an Early Stage of the Preprotein Import Process

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