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1.
Plant Physiol ; 187(4): 1916-1928, 2021 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-35235667

RESUMO

Insertion of membrane proteins into the lipid bilayer is a crucial step during their biosynthesis. Eukaryotic cells face many challenges in directing these proteins to their predestined target membrane. The hydrophobic signal peptide or transmembrane domain (TMD) of the nascent protein must be shielded from the aqueous cytosol and its target membrane identified followed by transport and insertion. Components that evolved to deal with each of these challenging steps range from chaperones to receptors, insertases, and sophisticated translocation complexes. One prominent translocation pathway for most proteins is the signal recognition particle (SRP)-dependent pathway which mediates co-translational translocation of proteins across or into the endoplasmic reticulum (ER) membrane. This textbook example of protein insertion is stretched to its limits when faced with secretory or membrane proteins that lack an amino-terminal signal sequence or TMD. Particularly, a large group of so-called tail-anchored (TA) proteins that harbor a single carboxy-terminal TMD require an alternative, post-translational insertion route into the ER membrane. In this review, we summarize the current research in TA protein insertion with a special focus on plants, address challenges, and highlight future research avenues.


Assuntos
Membrana Celular/metabolismo , Cloroplastos/metabolismo , Proteínas de Membrana/metabolismo , Redes e Vias Metabólicas , Mitocôndrias/metabolismo , Proteínas de Plantas/metabolismo , Transporte Proteico/efeitos dos fármacos
2.
J Cell Sci ; 131(10)2018 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-29661846

RESUMO

Tail-anchored (TA) proteins are anchored to their corresponding membrane via a single transmembrane segment (TMS) at their C-terminus. In yeast, the targeting of TA proteins to the endoplasmic reticulum (ER) can be mediated by the guided entry of TA proteins (GET) pathway, whereas it is not yet clear how mitochondrial TA proteins are targeted to their destination. It has been widely observed that some mitochondrial outer membrane (MOM) proteins are mistargeted to the ER when overexpressed or when their targeting signal is masked. However, the mechanism of this erroneous sorting is currently unknown. In this study, we demonstrate the involvement of the GET machinery in the mistargeting of suboptimal MOM proteins to the ER. These findings suggest that the GET machinery can, in principle, recognize and guide mitochondrial and non-canonical TA proteins. Hence, under normal conditions, an active mitochondrial targeting pathway must exist that dominates the kinetic competition against other pathways.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Adenosina Trifosfatases/metabolismo , Retículo Endoplasmático/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Retículo Endoplasmático/genética , Proteínas de Membrana/genética , Proteínas de Transporte da Membrana Mitocondrial/genética , Membranas Mitocondriais/metabolismo , Transporte Proteico , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
3.
Curr Biol ; 29(11): 1854-1865.e5, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31104938

RESUMO

Root hairs are tubular protrusions of the root epidermis that significantly enlarge the exploitable soil volume in the rhizosphere. Trichoblasts, the cell type responsible for root hair formation, switch from cell elongation to tip growth through polarization of the growth machinery to a predefined root hair initiation domain (RHID) at the plasma membrane. The emergence of this polar domain resembles the establishment of cell polarity in other eukaryotic systems [1-3]. Rho-type GTPases of plants (ROPs) are among the first molecular determinants of the RHID [4, 5], and later play a central role in polar growth [6]. Numerous studies have elucidated mechanisms that position the RHID in the cell [7-9] or regulate ROP activity [10-18]. The molecular players that target ROPs to the RHID and initiate outgrowth, however, have not been identified. We dissected the timing of the growth machinery assembly in polarizing hair cells and found that positioning of molecular players and outgrowth are temporally separate processes that are each controlled by specific ROP guanine nucleotide exchange factors (GEFs). A functional analysis of trichoblast-specific GEFs revealed GEF3 to be required for normal ROP polarization and thus efficient root hair emergence, whereas GEF4 predominantly regulates subsequent tip growth. Ectopic expression of GEF3 induced the formation of spatially confined, ROP-recruiting domains in other cell types, demonstrating the role of GEF3 to serve as a membrane landmark during cell polarization.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Raízes de Plantas/crescimento & desenvolvimento , Proteínas rho de Ligação ao GTP/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Raízes de Plantas/genética , Proteínas rho de Ligação ao GTP/metabolismo
4.
Methods Mol Biol ; 1691: 139-158, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29043675

RESUMO

Protein-protein interactions (PPIs) play vital roles in all subcellular processes and a number of tools have been developed for their detection and analysis. Each method has its unique set of benefits and drawbacks that need to be considered prior to their application. In fact, researchers are spoilt for choice when it comes to deciding which method to use for the initial detection of a PPI, and which to corroborate the findings. With constant improvements in microscope development, the possibilities of techniques to study PPIs in vivo, and in real time, are continuously enhanced, and expanded. Here, we describe three common approaches, their recent improvements incorporating a 2in1-cloning approach, and their application in plant cell biology: ratiometric Bimolecular Fluorescence Complementation (rBiFC), FRET Acceptor Photobleaching (FRET-AB), and Fluorescent Lifetime Imaging (FRET-FLIM), using Nicotiana benthamiana leaves and Arabidopsis thaliana cell culture protoplasts as transient expression systems.


Assuntos
Imagem Molecular , Proteínas de Plantas/metabolismo , Mapeamento de Interação de Proteínas/métodos , Expressão Gênica , Ordem dos Genes , Genes Reporter , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Processamento de Imagem Assistida por Computador , Microscopia Confocal/métodos , Imagem Molecular/métodos , Imagem Óptica/métodos , Protoplastos , Transfecção , Transformação Genética
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