An approach for reducing unwanted oligomerisation of DsRed fusion proteins

Gavin, Paul; Devenish, Rodney J.; Prescott, Mark

doi:10.1016/s0006-291x(02)02552-4

Cited by 34 publications

(34 citation statements)

References 21 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The sequence cassettes encoded the following components: (1) the RFP component retrieved by PCR from pQE31-DsRed.T3; 29 (2) the GFP component retrieved by PCR from pGEX-2 T carrying the ecliptic pHluorin; 30 and (3) the first 55 amino acids of the yeast citrate synthase precursor polypeptide amplified by PCR from yeast genomic DNA. 31 Visualization of mitochondria with mitochondrial matrix targeted GFP expressed from the constitutive TPI (triosephosphate isomerase) promoter was achieved by use of the plasmid pYX232-mtGFP. 18 In vivo labeling of mitochondrial translation products.…”

Section: Methodsmentioning

confidence: 99%

Mdm38 protein depletion causes loss of mitochondrial K+/H+ exchange activity, osmotic swelling and mitophagy

et al. 2007

View full text Add to dashboard Cite

Loss of the MDM38 gene product in yeast mitochondria results in a variety of phenotypic effects including reduced content of respiratory chain complexes, altered mitochondrial morphology and loss of mitochondrial K þ /H þ exchange activity resulting in osmotic swelling. By use of doxycycline-regulated shut-off of MDM38 gene expression, we show here that loss of K þ /H þ exchange activity and mitochondrial swelling are early events, associated with a reduction in membrane potential and fragmentation of the mitochondrial reticulum. Changes in the pattern of mitochondrially encoded proteins are likely to be secondary to the loss of K þ /H þ exchange activity. The use of a novel fluorescent biosensor directed to the mitochondrial matrix revealed that the loss of K þ /H þ exchange activity was immediately followed by morphological changes of mitochondria and vacuoles, the close association of these organelles and finally uptake of mitochondrial material by vacuoles. Nigericin, a K þ /H þ ionophore, fully prevented these effects of Mdm38p depletion. We conclude that osmotic swelling of mitochondria triggers selective mitochondrial autophagy or mitophagy.

show abstract

Section: Methodsmentioning

confidence: 99%

Mdm38 protein depletion causes loss of mitochondrial K+/H+ exchange activity, osmotic swelling and mitophagy

et al. 2007

View full text Add to dashboard Cite

show abstract

“…YRD15 (MATα,his3,ura3,leu2,[rho + ]) was the parental strain (Straffon et al, 1998). Strains lacking endogenous subunit γ and expressing γ-DsRed and γ-GFP fusions have been described elsewhere (Gavin et al, 2002;Prescott et al, 2003). The chromosomal ATP3 gene encoding the γ subunit was modified in strain γ-27-DsRed to express the DsRed protein (Clontech, Palo Alto, CA) fused to the Cterminus of subunit γ via a 27 amino acid linker.…”

Section: Yeast Strains and Growth Conditionsmentioning

confidence: 99%

“…By contrast, expression of an equivalent subunit γ-GFP fusion did not impair growth of cells, or result in mtATPase oligomerisation (Gavin et al, 2002;Prescott et al, 2003). DsRed is known to form tetramers in vitro and in living cells (Baird et al, 2000), a property that can cause oligomerisation of proteins to which it is fused (Gavin et al, 2002;Lauf et al, 2001;Mizuno et al, 2001). Thus, we concluded that strong interactions between the DsRed moieties of individual mtATPase complexes incorporating the γ-DsRed fusion led to stable associations between multiple adjacent mtATPase complexes.…”

mentioning

confidence: 95%

“…Recently we found that respiratory growth of yeast cells having mtATPase incorporating a subunit γ-DsRed fusion was impaired relative to control cells (Gavin et al, 2002).…”

mentioning

confidence: 98%

“…Thus, we concluded that strong interactions between the DsRed moieties of individual mtATPase complexes incorporating the γ-DsRed fusion led to stable associations between multiple adjacent mtATPase complexes. Co-expression of DsRed not fused to another protein (free DsRed) that was targeted to mitochondria partially reversed the slower growth phenotype of cells, presumably because the free DsRed competed with interaction sites on the subunit γ-DsRed fusion proteins, thereby releasing some mtATPase complexes from their oligomeric arrangement (Gavin et al, 2002).…”

mentioning

confidence: 99%

See 2 more Smart Citations

Cross-linking ATP synthase complexes in vivo eliminates mitochondrial cristae

Gavin

Prescott

Luff

et al. 2004

Journal of Cell Science

Self Cite

View full text Add to dashboard Cite

We have used the tetrameric nature of the fluorescent protein DsRed to cross-link F1FO-ATPase complexes incorporating a subunit γ-DsRed fusion protein in vivo. Cells expressing such a fusion protein have impaired growth relative to control cells. Strikingly, fluorescence microscopy of these cells revealed aberrant mitochondrial morphology. Electron microscopy of cell sections revealed the absence of cristae and multiple layers of unfolded inner mitochondrial membrane. Complexes recovered from detergent lysates of mitochondria were present largely as tetramers. Co-expression of `free' DsRed targeted to the mitochondria reduced F1FO-ATPase oligomerisation and partially reversed the impaired growth and abnormal mitochondrial morphology. We conclude that the correct arrangement of F1FO-ATPase complexes within the mitochondrial inner membrane is crucial for the genesis and/or maintenance of mitochondrial cristae and morphology. Our findings further suggest that F1FO-ATPase can exist in oligomeric associations within the membrane during respiratory growth.

show abstract