Limits...
Mitochondrial outer and inner membrane fusion requires a modified carrier protein.

Hoppins S, Horner J, Song C, McCaffery JM, Nunnari J - J. Cell Biol. (2009)

Bottom Line: Fzo1 and Mgm1 are conserved guanosine triphosphatases that reside in the outer and inner membranes, respectively.At each membrane, these conserved proteins are required for the distinct steps of membrane tethering and lipid mixing.The third essential component is Ugo1, an outer membrane protein in the mitochondrial transport protein family.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, University of California, Davis, CA 95616, USA.

ABSTRACT
In yeast, three proteins are essential for mitochondrial fusion. Fzo1 and Mgm1 are conserved guanosine triphosphatases that reside in the outer and inner membranes, respectively. At each membrane, these conserved proteins are required for the distinct steps of membrane tethering and lipid mixing. The third essential component is Ugo1, an outer membrane protein in the mitochondrial transport protein family. We show that Ugo1 is a modified member of this family, containing three transmembrane domains and existing as a dimer, a structure that is critical for the fusion function of Ugo1. Our functional analysis of Ugo1 indicates that it is required distinctly for both outer and inner membrane fusion after membrane tethering, indicating that it operates at the lipid-mixing step of fusion. This role is distinct from the fusion dynamin-related proteins and thus demonstrates that at each membrane, a single fusion protein is not sufficient to drive the lipid-mixing step, but instead, this step requires a more complex assembly of proteins.

Show MeSH

Related in: MedlinePlus

Characterization of temperature-sensitive alleles of Ugo1. (A) Wild-type (UGO1 and UGO1-HA) and ugo1 temperature-sensitive cells (ugo1-1–ugo1-5) were plated on YPD and YPEG plates and incubated at the permissive (23°C) or nonpermissive temperature (37°C). (B, top) A schematic representation of the in vitro inner membrane fusion assay. (middle) Fluorescent images of in vitro mitochondrial fusion reactions with mitochondria isolated from UGO1 and ugo1-1. Arrows indicate fusion events. (bottom) A comparison of mitochondrial in vitro fusion efficiency in UGO1, ugots, fzo1-1, and mgm1-5 mitochondria at the permissive (23°C) and nonpermissive temperature (37°C). Fusion efficiency of ugo1ts mitochondria is expressed as a percentage of wild-type fusion, which averaged 8.5% of total mitochondria. Data are represented as mean ± SEM. Bars, 1 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC2654124&req=5

fig4: Characterization of temperature-sensitive alleles of Ugo1. (A) Wild-type (UGO1 and UGO1-HA) and ugo1 temperature-sensitive cells (ugo1-1–ugo1-5) were plated on YPD and YPEG plates and incubated at the permissive (23°C) or nonpermissive temperature (37°C). (B, top) A schematic representation of the in vitro inner membrane fusion assay. (middle) Fluorescent images of in vitro mitochondrial fusion reactions with mitochondria isolated from UGO1 and ugo1-1. Arrows indicate fusion events. (bottom) A comparison of mitochondrial in vitro fusion efficiency in UGO1, ugots, fzo1-1, and mgm1-5 mitochondria at the permissive (23°C) and nonpermissive temperature (37°C). Fusion efficiency of ugo1ts mitochondria is expressed as a percentage of wild-type fusion, which averaged 8.5% of total mitochondria. Data are represented as mean ± SEM. Bars, 1 µm.

Mentions: To determine whether Ugo1 plays a direct role in mitochondrial fusion, conditional temperature-sensitive alleles of UGO1 were isolated. To create and screen for ugo1ts alleles, we amplified the UGO1-HA gene by PCR under low fidelity DNA polymerase conditions and integrated mutagenized ugo1-HA into the genome of the wild-type yeast strain W303 at the UGO1 locus. To identify ugo1-HAts strains, transformants were screened for specific growth defects on the fermentable carbon source glycerol at 37°C. Five independent ugo1-HAts strains, displaying the largest differences in growth rate on glycerol at permissive and nonpermissive temperatures, were chosen for further characterization (Fig. 4 A, ugo1-1–ugo1-5). We performed sequence analysis of the UGO1 locus in each of these strains and discovered that each ugo1 allele contains between four and six missense mutations (Table I). Western analysis of SDS-PAGE of total cellular extracts or isolated mitochondria from each ugo1-HAts strain using either anti-HA or anti-Ugo1 polyclonal antibodies indicated that Ugo1 is expressed at levels comparable with wild type in each ugo1 mutant (unpublished data). In addition, analysis of mitochondrial morphology using mitochondria GFP in ugo1ts strains indicated that at the permissive temperature mitochondrial morphology was reticular, which is similar to that observed in UGO1-HA control cells (unpublished data). Upon shifting to the nonpermissive temperature, ugo1ts cells contained mitochondria that were fragmented compared with UGO1-HA control cells, which is consistent with the reported essential role of Ugo1 in mitochondrial fusion (Sesaki and Jensen, 2001; and unpublished data).


Mitochondrial outer and inner membrane fusion requires a modified carrier protein.

Hoppins S, Horner J, Song C, McCaffery JM, Nunnari J - J. Cell Biol. (2009)

Characterization of temperature-sensitive alleles of Ugo1. (A) Wild-type (UGO1 and UGO1-HA) and ugo1 temperature-sensitive cells (ugo1-1–ugo1-5) were plated on YPD and YPEG plates and incubated at the permissive (23°C) or nonpermissive temperature (37°C). (B, top) A schematic representation of the in vitro inner membrane fusion assay. (middle) Fluorescent images of in vitro mitochondrial fusion reactions with mitochondria isolated from UGO1 and ugo1-1. Arrows indicate fusion events. (bottom) A comparison of mitochondrial in vitro fusion efficiency in UGO1, ugots, fzo1-1, and mgm1-5 mitochondria at the permissive (23°C) and nonpermissive temperature (37°C). Fusion efficiency of ugo1ts mitochondria is expressed as a percentage of wild-type fusion, which averaged 8.5% of total mitochondria. Data are represented as mean ± SEM. Bars, 1 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2654124&req=5

fig4: Characterization of temperature-sensitive alleles of Ugo1. (A) Wild-type (UGO1 and UGO1-HA) and ugo1 temperature-sensitive cells (ugo1-1–ugo1-5) were plated on YPD and YPEG plates and incubated at the permissive (23°C) or nonpermissive temperature (37°C). (B, top) A schematic representation of the in vitro inner membrane fusion assay. (middle) Fluorescent images of in vitro mitochondrial fusion reactions with mitochondria isolated from UGO1 and ugo1-1. Arrows indicate fusion events. (bottom) A comparison of mitochondrial in vitro fusion efficiency in UGO1, ugots, fzo1-1, and mgm1-5 mitochondria at the permissive (23°C) and nonpermissive temperature (37°C). Fusion efficiency of ugo1ts mitochondria is expressed as a percentage of wild-type fusion, which averaged 8.5% of total mitochondria. Data are represented as mean ± SEM. Bars, 1 µm.
Mentions: To determine whether Ugo1 plays a direct role in mitochondrial fusion, conditional temperature-sensitive alleles of UGO1 were isolated. To create and screen for ugo1ts alleles, we amplified the UGO1-HA gene by PCR under low fidelity DNA polymerase conditions and integrated mutagenized ugo1-HA into the genome of the wild-type yeast strain W303 at the UGO1 locus. To identify ugo1-HAts strains, transformants were screened for specific growth defects on the fermentable carbon source glycerol at 37°C. Five independent ugo1-HAts strains, displaying the largest differences in growth rate on glycerol at permissive and nonpermissive temperatures, were chosen for further characterization (Fig. 4 A, ugo1-1–ugo1-5). We performed sequence analysis of the UGO1 locus in each of these strains and discovered that each ugo1 allele contains between four and six missense mutations (Table I). Western analysis of SDS-PAGE of total cellular extracts or isolated mitochondria from each ugo1-HAts strain using either anti-HA or anti-Ugo1 polyclonal antibodies indicated that Ugo1 is expressed at levels comparable with wild type in each ugo1 mutant (unpublished data). In addition, analysis of mitochondrial morphology using mitochondria GFP in ugo1ts strains indicated that at the permissive temperature mitochondrial morphology was reticular, which is similar to that observed in UGO1-HA control cells (unpublished data). Upon shifting to the nonpermissive temperature, ugo1ts cells contained mitochondria that were fragmented compared with UGO1-HA control cells, which is consistent with the reported essential role of Ugo1 in mitochondrial fusion (Sesaki and Jensen, 2001; and unpublished data).

Bottom Line: Fzo1 and Mgm1 are conserved guanosine triphosphatases that reside in the outer and inner membranes, respectively.At each membrane, these conserved proteins are required for the distinct steps of membrane tethering and lipid mixing.The third essential component is Ugo1, an outer membrane protein in the mitochondrial transport protein family.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, University of California, Davis, CA 95616, USA.

ABSTRACT
In yeast, three proteins are essential for mitochondrial fusion. Fzo1 and Mgm1 are conserved guanosine triphosphatases that reside in the outer and inner membranes, respectively. At each membrane, these conserved proteins are required for the distinct steps of membrane tethering and lipid mixing. The third essential component is Ugo1, an outer membrane protein in the mitochondrial transport protein family. We show that Ugo1 is a modified member of this family, containing three transmembrane domains and existing as a dimer, a structure that is critical for the fusion function of Ugo1. Our functional analysis of Ugo1 indicates that it is required distinctly for both outer and inner membrane fusion after membrane tethering, indicating that it operates at the lipid-mixing step of fusion. This role is distinct from the fusion dynamin-related proteins and thus demonstrates that at each membrane, a single fusion protein is not sufficient to drive the lipid-mixing step, but instead, this step requires a more complex assembly of proteins.

Show MeSH
Related in: MedlinePlus