A tag at the carboxy terminus prevents membrane integration of VDAC1 in mammalian mitochondria.
beta-Barrel proteins are found in the outer membranes of bacteria, chloroplasts and mitochondria. The evolutionary conserved sorting and assembly machinery (SAM complex) assembles mitochondrial beta-barrel proteins, such as voltage-dependent anion-selective channel 1 (VDAC1), into complexes in the outer membrane by recognizing a sorting beta-signal in the carboxy-terminal part of the protein. Here we show that in mammalian mitochondria, masking of the C-terminus of beta-barrel proteins by a tag leads to accumulation of soluble misassembled protein in the intermembrane space, which causes mitochondrial fragmentation and loss of membrane potential. A similar phenotype is observed if the beta-signal is shortened, removed or when the conserved hydrophobic residues in the beta-signal are mutated. The length of the tag at the C-terminus is critical for the assembly of VDAC1, as well as the amino acid residues at positions 130, 222, 225 and 251 of the protein. We propose that if the recognition of the beta-signal or the folding of the beta-barrel proteins is inhibited, the nonassembled protein will accumulate in the intermembrane space, aggregate and damage mitochondria. This effect offers easy tools for studying the requirements for the membrane assembly of beta-barrel proteins, but also advises caution when interpreting the outcome of the beta-barrel protein overexpression experiments.
Kozjak-Pavlovic
Vera
V
Department of Molecular Biology, Max Planck Institute for Infection Biology, Charitéplatz 1, 10117 Berlin, Germany.
Ross
Katharina
K
Götz
Monika
M
Goosmann
Christian
C
Rudel
Thomas
T
D000595:Amino Acid Sequence; D006367:HeLa Cells; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D053078:Membrane Potential, Mitochondrial; D008928:Mitochondria / Q000378:metabolism* / Q000648:ultrastructure; D051336:Mitochondrial Membranes / Q000378:metabolism* / Q000648:ultrastructure; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D017433:Protein Structure, Secondary; D021381:Protein Transport; D013329:Structure-Activity Relationship; D050995:Voltage-Dependent Anion Channel 1 / Q000737:chemistry* / Q000378:metabolism* / Q000633:toxicity
D050995:Voltage-Dependent Anion Channel 1