Intracellular vesicle trafficking plays an essential role in mitochondrial quality control.

The gene products Bet1, Slh, and CG10144, predicted to function in intracellular vesicle trafficking, were previously found to be essential for mitochondrial nucleoid maintenance. Here we show that Slh and Bet1 cooperate to maintain mitochondrial functions. In their absence, mitochondrial content, membrane potential, and respiration became abnormal, accompanied by mitochondrial proteotoxic stress, but without direct effects on mtDNA. Immunocytochemistry showed that both Slh and Bet1 are localized at the Golgi, together with a proportion of Rab5-positive vesicles. Some Bet1, as well as a tiny amount of Slh, cofractionated with highly purified mitochondria, while live-cell imaging showed coincidence of fluorescently tagged Bet1 with most Lysotracker-positive and a small proportion of Mitotracker-positive structures. This three-way association was disrupted in cells knocked down for Slh, although colocalized lysosomal and mitochondrial signals were still seen. Neither Slh nor Bet1 was required for global mitophagy or endocytosis, but prolonged Slh knockdown resulted in G2 growth arrest, with increased cell diameter. These effects were shared with knockdown of betaCOP but not of CG1044, Snap24, or Syntaxin6. Our findings implicate vesicle sorting at the -Golgi in mitochondrial quality control.