Assessment of the integral membrane protein topology in living cells

Zamyatnin, Andrey A.; Solovyev, Andrey G.; Bozhkov, Peter V.; Valkonen, Jari P. T.; Morozov, Sergey Y.; Savenkov, Eugene I.

doi:10.1111/j.1365-313x.2006.02674.x

Cited by 103 publications

(107 citation statements)

References 40 publications

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“…However, highly specific interactions between membrane proteins have been demonstrated using this technique in other systems (26)(27)(28). Also, the absence of an interaction with the plasma membrane protein PIP2-1 makes this possibility unlikely.…”

Section: Discussionmentioning

confidence: 99%

Organization of cellulose synthase complexes involved in primary cell wall synthesis in Arabidopsis thaliana

Desprez

Juraniec

Crowell

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

528

605

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In all land plants, cellulose is synthesized from hexameric plasma membrane complexes. Indirect evidence suggests that in vascular plants the complexes involved in primary wall synthesis contain three distinct cellulose synthase catalytic subunits (CESAs). In this study, we show that CESA3 and CESA6 fused to GFP are expressed in the same cells and at the same time in the hypocotyl of etiolated seedlings and migrate with comparable velocities along linear trajectories at the cell surface. We also show that CESA3 and CESA6 can be coimmunoprecipitated from detergent-solubilized extracts, their protein levels decrease in mutants for either CESA3, CESA6, or CESA1 and CESA3, CESA6 and also CESA1 can physically interact in vivo as shown by bimolecular fluorescence complementation. We also demonstrate that CESA6-related CESA5 and CESA2 are partially, but not completely, redundant with CESA6 and most likely compete with CESA6 for the same position in the cellulose synthesis complex. Using promoter-␤-glucuronidase fusions we show that CESA5, CESA6, and CESA2 have distinct overlapping expression patterns in hypocotyl and root corresponding to different stages of cellular development. Together, these data provide evidence for the existence of binding sites for three distinct CESA subunits in primary wall cellulose synthase complexes, with two positions being invariably occupied by CESA1 and CESA3, whereas at least three isoforms compete for the third position. Participation of the latter three isoforms might fine-tune the CESA complexes for the deposition of microfibrils at distinct cellular growth stages. C ellulose microfibrils are synthesized from a multiprotein complex inserted into the plasma membrane. These ''rosette'' complexes consist of six globules, each of which contains multiple cellulose synthase catalytic subunits (CESAs). These complexes migrate in the plasma membrane along microtubules, propelled by the polymerization of the ␤-1,4-glucan chains (1).Plant CESA genes are members of multigene families. Arabidopsis has 10 CESA isoforms that, based on sequence comparison with other plant species, can be classified into six orthologous groups (2). Mutational analysis shows that these six groups of isoforms have nonredundant functions in cellulose synthesis. Mutants for three isoforms (CESA4, CESA7, and CESA8) show defects in cellulose synthesis specifically in secondary walls (3)(4)(5). Microarray data show that the mRNAs for the three genes are coregulated (6, 7). The three proteins are expressed in the same cell types during secondary cell wall deposition, and co-immunoprecipitation (IP) experiments show that all three proteins interact (3). Although the interactions remain to be validated in vivo, these data strongly suggest that at least in these cells the complexes contain three isoforms. Mutants for isoforms CESA1, CESA3, and CESA6 have cellulose defects in primary cell walls (8-11). The three genes are also coregulated at the mRNA level (12). It is not known, however, whether the corresponding proteins are ...

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Section: Discussionmentioning

confidence: 99%

Organization of cellulose synthase complexes involved in primary cell wall synthesis in Arabidopsis thaliana

Desprez

Juraniec

Crowell

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

528

605

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“…Most BiMC studies have focused on soluble proteins, although several have examined interactions of integral membrane proteins (30)(31)(32). One problem that is mentioned in these reports is where to place the tag.…”

Section: Discussionmentioning

confidence: 99%

Bimolecular complementation reveals that glycoproteins gB and gH/gL of herpes simplex virus interact with each other during cell fusion

Atanasiu

Whitbeck

Cairns

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

163

205

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Herpes simplex virus entry into cells requires four glycoproteins, gB, gD, gH, and gL. Binding of gD to one of its receptors triggers steps requiring the core fusion proteins, gB and the gH/gL heterodimer. There is evidence that gH/gL initiates hemifusion of cells, but whether this complex interacts physically with gB to cause complete fusion is unknown. We used bimolecular complementation (BiMC) of enhanced yellow fluorescent protein (EYFP) to detect glycoprotein interactions during cell-cell fusion. The N-or C-terminal half of EYFP was fused to the C terminus of gD, gB, and gH to form six chimeric proteins (Dn, Dc, Bn, Bc, Hn, and Hc). BiMC was detected by confocal microscopy. Receptor-bearing (C10) cells cotransfected with Dn and Bc or Dn, Hc, and untagged gL exhibited EYFP fluorescence, indicative of interactions between gD and gB and between gD and gH/gL. EYFP complementation did not occur in cells transfected with gL, Bc, and Hn. However, when gD was coexpressed with these other three proteins, cell-cell fusion occurred and the syncytia exhibited bright EYFP fluorescence. To separate glycoprotein expression from fusion, we transfected C10 cells with gL, Bc, and Hn for 20 h and then added soluble gD to trigger fusion. We detected fluorescent syncytia within 10 min, and both their number and size increased with exposure time to gD. Thus, when gD binds its receptor, the core fusion machinery is triggered to form a multiprotein complex as a step in fusion and possibly virus entry.BiMC ͉ fusion ͉ HSV ͉ interaction ͉ EYFP

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“…from cell transformation to microscopy analyses), BiFC may introduce elements that lead to events that do not reflect normal cell physiology and these must be considered for correct interpretation of the results. Another point to consider is that in conditions of elevated availability of the YFP halves, YFP molecules may be generated simply because YFP halves tend to associate (Zamyatnin et al, 2006). It has also been reported that overexpression in mam- malian cells of N-and C-terminal YFP peptides alone resulted in a low fluorescence emission signal; thus, the levels of expression of the split-YFP halves may be a factor to consider in BiFC experiments (Ozalp et al, 2005).…”

Section: Split Yfpmentioning

confidence: 99%

“…Although this method is seemingly powerful, it may require careful controls to ensure that the disappearance of the fluorescence signal is not due to technical issues, such as photobleaching or excessive membrane permeabilization that may drastically alter the cell environment. Recently, Zamyatnin and colleagues demonstrated the usefulness of BiFC to determine membrane protein topology; model proteins used were the beet yellows virus-encoded p6 membrane-embedded movement protein, a protein with known topology, and the potato mop-top virus-encoded integral membrane TGBp2 protein with predicted topology (Zamyatnin et al, 2006). This application is based on the property of free split-YFP fragments to associate in plant cells as a result of their overexpression from strong promoters (Walter et al, 2004).…”

Section: Split Yfpmentioning

confidence: 99%

Advances in Fluorescent Protein-Based Imaging for the Analysis of Plant Endomembranes

2008

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An exciting new era for live cell imaging of plant endomembranes was ushered in just over 10 years ago when Jim Haseloff and colleagues reported on the removal of a cryptic intron in the sequence of wildtype GFP for successful expression in plant cells (Haseloff et al., 1997). Haseloff's success was quickly welcomed by labs around the globe; by targeting GFP to secretory organelles, scientists could now bring to light the secret life of plant endomembranes. The plant endomembranes comprise several organelles functionally interlinked for the production and transport of secretory compounds, such as proteins, lipids, and sugars. Most of the secretory compounds are synthesized in the endoplasmic reticulum (ER) and then transported to the Golgi apparatus to be sorted for transport to distal compartments such as the plasma membrane and vacuoles. A forward transport of secretory molecules from the ER is counterbalanced by a retrograde flow from distal compartments that allows membrane homeostasis and communication with the cell's surroundings. Fluorescent protein technology applied to live cell imaging of plant endomembranes has aided immensely in providing subcellular markers for the study of the complex spatial and temporal relationships among secretory organelles. Live cell imaging is now moving forward to novel challenges. With the integration of genomic, transcriptomic, and proteomic techniques, we begin to collect data on the putative functions of plant genes; we need to understand the intricate relationships among thousands of proteins. To complete the puzzle, we must understand how the pieces fit together; we must define the functions of gene products. Important questions include: When are our proteins synthesized? Where are they targeted? With what elements do they interact? Advances in the development of optical imaging at the cellular level now offer the exciting opportunity to answer these questions.In this Update, we discuss several state-of-the-art applications of GFP imaging and how these techniques have been used to answer critical biological questions, with a focus on plant endomembrane trafficking.

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Assessment of the integral membrane protein topology in living cells

Cited by 103 publications

References 40 publications

Organization of cellulose synthase complexes involved in primary cell wall synthesis in Arabidopsis thaliana

Organization of cellulose synthase complexes involved in primary cell wall synthesis in Arabidopsis thaliana

Bimolecular complementation reveals that glycoproteins gB and gH/gL of herpes simplex virus interact with each other during cell fusion

Advances in Fluorescent Protein-Based Imaging for the Analysis of Plant Endomembranes

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