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Targeting and function of CAH1: Characterization of a novel protein pathway to the plant cell chloroplast
Umeå University, Faculty of Science and Technology, Plant Physiology. (Göran Samuelsson)
2010 (English)Doctoral thesis, comprehensive summary (Other academic)Alternative title
Transport och funktion av CAH1 : Karakterisering av en ny transportväg för proteiner till växtcellens kloroplast (Swedish)
Abstract [en]

The chloroplast is the organelle within a plant cell where photosynthesis takes place. This organelle originates from a cyanobacterium that was engulfed by a eukaryotic cell. During the transition from endosymbiont to organelle most of the cyanobacterial genes were transferred to the nuclear genome of the host cell, resulting in a chloroplast with a much reduced genome that requires massive import of gene products (proteins) back to the organelle. The majority of these proteins are translated in the cytosol as pre-proteins containing targeting information that directs them to a translocon complex in the chloroplast envelope, the Toc-Tic system, through which these proteins are transported.

We have identified a protein in the model plant Arabidopsis thaliana, CAH1, that is trafficked via the endomembrane system (ER/Golgi apparatus) to the chloroplast instead of using the Toc-Tic machinery. This transport is partly mediated by canonical vesicle trafficking elements involved in ER to Golgi transport, such as Sar1 and RabD GTPases. Analysis of point mutated variants of CAH1 showed that both N-linked glycans and an intra-molecular disulphide bridge are required for correct folding, trafficking and function of the protein. Since chloroplasts lack N-glycosylation machinery, we propose that a route for chloroplast proteins that require endomembrane-specific post-translational modifications for their functionality exists as a complement to the Toc-Tic system.

We also show that mutant plants with disrupted CAH1 gene expression have reduced rates of CO2 uptake and accumulate lower amounts of starch compared to wild-type plants, indicating an important function of the CAH1 protein for the photosynthetic capacity of Arabidopsis.

Further study of CAH1 will not only be important to reveal its role in photosynthesis, but characterization of this novel protein pathway to the chloroplast can also shed light on how the plant cell evolved and clarify the purpose of keeping several chloroplast import pathways working in parallel. In addition, knowledge about this pathway could increase the opportunities for using plants as bio-factories for production of recombinant glycoproteins, which make up the vast majority of the bio-pharmaceutical molecules.

Abstract [sv]

Kloroplasten är den organell i växtcellen där fotosyntesen sker. Denna organell härstammar från en cyanobakterie som togs upp av en eukaryot cell. Under omvandlingen från endosymbiont till organell har de flesta av den ursprungliga cyanobakteriens gener flyttats över till växtcellens eget kärngenom, vilket resulterat i en kloroplast som endast kan producera ett fåtal av de proteiner den behöver och som istället kräver att en mängd genprodukter (proteiner) transporteras tillbaka till organellen. De flesta av dessa proteiner syntetiseras i cytosolen som polypeptider innehållande en speciell signal för kloroplasten, och tranporteras över kloroplastens dubbelmembran (envelop) med hjälp av ett specifikt importsystem (Toc-Tic).

Vi har identifierat ett protein i modellväxten Arabidopsis thaliana (CAH1) som istället för att använda Toc-Tic tranporteras via det endomembrana systemet (ER/Golgi). Transporten sker delvis med hjälp av faktorer involverade i normal vesikeltransport, t.ex. Sar1 och RabD GTPaser (mellan ER och Golgi). Genom att uttycka och analysera punktmuterade varianter av CAH1 har vi kunnat visa att både sockergrupper kopplade till proteinet, samt en intern svavelbrygga, är nödvändiga för korrekt veckning, transport och funktion av proteinet. Då kloroplasten saknar eget maskineri för att koppla sådana sockergrupper till proteiner så föreslår vi att anledningen till att denna rutt existerar, som ett komplement till Toc-Tic, är för att proteiner beroende av denna typ av modifiering ska kunna finnas i kloroplasten.

Vi visar också att muterade växter som inte kan uttrycka genen som kodar för CAH1 uppvisar lägre upptag av CO2, samt ackumulerar mindre stärkelse än vildtypplantor, vilket antyder att CAH1 har en viktig funktion för den fotosyntetiska förmågan hos Arabidopsis.

För att kunna fastställa den exakta funktionen för CAH1 kommer ytterliga studier att vara nödvändiga. En fördjupad karaktärisering av transportvägen som CAH1 följer till kloroplasten kan dessutom ge kunskap om hur växtcellen uppkom, samt besvara varför flera importvägar arbetar till synes parallellt med varandra. Kunskap om denna transportväg kan även bidra med användbar information i försöken att nyttja växter till att uttrycka rekombinanta N-glykosylerade proteiner, t. ex. antikroppar och vacciner.

Place, publisher, year, edition, pages
Umeå: Institutionen för fysiologisk botanik, Umeå universitet , 2010. , 68 p.
Keyword [en]
Arabidopsis, chloroplast, endomembrane system, CAH1, protein targeting, N-glycosylation
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-30509ISBN: 978-91-7264-933-0 (print)OAI: oai:DiVA.org:umu-30509DiVA: diva2:284338
Public defence
2010-01-29, KBC-huset, KB3A9, Umeå Universitet, Umeå, 10:00 (English)
Opponent
Supervisors
Available from: 2010-01-08 Created: 2010-01-05 Last updated: 2010-01-08Bibliographically approved
List of papers
1. Evidence for a protein transported through the secretory pathway en route to the higher plant chloroplast.
Open this publication in new window or tab >>Evidence for a protein transported through the secretory pathway en route to the higher plant chloroplast.
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2005 (English)In: Nature Cell Biology, ISSN 1465-7392, Vol. 7, no 12, 1224-31 p.Article in journal (Refereed) Published
Abstract [en]

In contrast to animal and fungal cells, green plant cells contain one or multiple chloroplasts, the organelle(s) in which photosynthetic reactions take place. Chloroplasts are believed to have originated from an endosymbiotic event and contain DNA that codes for some of their proteins. Most chloroplast proteins are encoded by the nuclear genome and imported with the help of sorting signals that are intrinsic parts of the polypeptides. Here, we show that a chloroplast-located protein in higher plants takes an alternative route through the secretory pathway, and becomes N-glycosylated before entering the chloroplast.

Keyword
Arabidopsis/metabolism/*ultrastructure, Arabidopsis Proteins/metabolism, Carbonic Anhydrases/genetics, Chloroplasts/*metabolism, Cytoplasmic Vesicles/metabolism, Glycosylation, Green Fluorescent Proteins/genetics, Microscopy; Fluorescence, Protein Sorting Signals, Protein Transport, Transfection
Identifiers
urn:nbn:se:umu:diva-10643 (URN)doi:10.1038/ncb1330 (DOI)16284624 (PubMedID)
Available from: 2007-05-23 Created: 2007-05-23 Last updated: 2015-05-07Bibliographically approved
2. Optimization of the 2A peptide coexpression system to study trafficking of the plastid N-glycoprotein CAH1 in Arabidopsis thaliana
Open this publication in new window or tab >>Optimization of the 2A peptide coexpression system to study trafficking of the plastid N-glycoprotein CAH1 in Arabidopsis thaliana
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(English)Manuscript (preprint) (Other academic)
Keyword
2A peptide, Arabidopsis, CAH1, chloroplast, endoplasmic reticulum, ER-toGolgi transport, Golgi apparatus, small GTPases, protein transport
Identifiers
urn:nbn:se:umu:diva-30505 (URN)
Available from: 2010-01-05 Created: 2010-01-05 Last updated: 2015-04-29
3. N-glycosylation is required for trafficking and activity of a chloroplast-localized carbonic anhydrase (CAH1) in Arabidopsis thaliana
Open this publication in new window or tab >>N-glycosylation is required for trafficking and activity of a chloroplast-localized carbonic anhydrase (CAH1) in Arabidopsis thaliana
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(English)Manuscript (preprint) (Other academic)
Identifiers
urn:nbn:se:umu:diva-30506 (URN)
Available from: 2010-01-05 Created: 2010-01-05 Last updated: 2015-04-29
4. Specific suppression of the chloroplast N-glycosylated carbonic anhydrase (CAH1) has major impact on the photosynthetic performance of Arabidopsis thaliana
Open this publication in new window or tab >>Specific suppression of the chloroplast N-glycosylated carbonic anhydrase (CAH1) has major impact on the photosynthetic performance of Arabidopsis thaliana
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(English)Manuscript (preprint) (Other academic)
Identifiers
urn:nbn:se:umu:diva-30507 (URN)
Available from: 2010-01-05 Created: 2010-01-05 Last updated: 2015-04-29

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