MitoNEET (mNEET) is a dimeric mitochondrial outer membrane protein implicated in many facets of human pathophysiology, notably diabetes and cancer, but its molecular function remains poorly characterized. In this study, we generated and analyzed mNEET KO cells and found that in these cells the mitochondrial network was disturbed. Analysis of 3D-EM reconstructions and of thin sections revealed that genetic inactivation of mNEET did not affect the size of mitochondria but that the frequency of intermitochondrial junctions was reduced. Loss of mNEET decreased cellular respiration, because of a reduction in the total cellular mitochondrial volume, suggesting that intermitochondrial contacts stabilize individual mitochondria. Reexpression of mNEET in mNEET KO cells restored the WT morphology of the mitochondrial network, and reexpression of a mutant mNEET resistant to oxidative stress increased in addition the resistance of the mitochondrial network to H 2 O 2 -induced fragmentation. Finally, overexpression of mNEET increased strongly intermitochondrial contacts and resulted in the clustering of mitochondria. Our results suggest that mNEET plays a specific role in the formation of intermitochondrial junctions and thus participates in the adaptation of cells to physiological changes and to the control of mitochondrial homeostasis.mitoNEET | CISD1 | mitochondria | intermitochondrial junctions | endoplasmic reticulum

TM9 family proteins (also named Phg1 proteins) have been previously shown to control cell adhesion by determining the cell surface localization of adhesion proteins such as the Dictyostelium SibA protein. Here, we show that the glycine-rich transmembrane domain (TMD) of SibA is sufficient to confer Phg1A-dependent surface targeting to a reporter protein. Accordingly, in Dictyostelium phg1A-knockout (KO) cells, proteins with glycine-rich TMDs were less efficiently transported out of the endoplasmic reticulum (ER) and to the cell surface. Phg1A, as well as its human ortholog TM9SF4 specifically associated with glycine-rich TMDs. In human cells, genetic inactivation of TM9SF4 resulted in an increased retention of glycine-rich TMDs in the endoplasmic reticulum, whereas TM9SF4 overexpression enhanced their surface localization. The bulk of the TM9SF4 protein was localized in the Golgi complex and a proximity-ligation assay suggested that it might interact with glycine-rich TMDs. Taken together, these results suggest that one of the main roles of TM9 proteins is to serve as intramembrane cargo receptors controlling exocytosis and surface localization of a subset of membrane proteins.

Previous studies have shown that TM9SF4 interacts with glycine-rich transmembrane domains (TMDs) and promotes their surface localization, presumably by escorting them along the secretory pathway. Here, we delineated the role of TM9 proteins in the sorting of TMDs. Our results indicate that TM9SF4 interacts with and sorts a variety of TMDs. In human embryonic kidney (HEK) cells, a TMD carrying a positively charged residue (T-R1) or a negatively charged residue (T-D1) was localized to the endoplasmic reticulum (ER), but partially relocated to the Golgi complex upon overexpression of TM9SF4. These results show that TM9SF4 controls the sorting of TMDs at the ER-Golgi interface. Remarkably, sorting of T-R1 in HCT116 cells was different from that in HEK cells: in HCT116 cells, a substantial fraction of T-R1 was localized to the Golgi complex, and it was relocated to the ER by genetic ablation of TM9SF4. This observation indicates that TM9SF4 sorting activity differs in HEK and HCT116 cells, resulting in different sorting of TMDs in these two cell types. Although TM9SF1 associated with several TMDs, it did not visibly alter their intracellular transport in the secretory pathway and may function in other intracellular transport pathways.

BackgroundImmunolocalization of cellular antigens typically requires fixation and permeabilization of cells, prior to incubation with antibodies.FindingsAssessing a test protein abundantly present at the cell surface of Dictyostelium cells, we show that in fixed cells, permeabilization extracts almost completely this cell surface antigen. The extent of this artifact is variable depending on the procedure used for labeling and permeabilization, as well as on the antigen considered.ConclusionsAn optimized protocol for labeling both surface and intracellular antigens without significant loss of labeling is proposed.

BackgroundThe envelope protein of lentiviruses are type I transmembrane proteins, and their transmembrane domain contains conserved potentially charged residues. This highly unusual feature would be expected to cause endoplasmic reticulum (ER) localization. The aim of this study was to determine by which means the HIV-1 Env protein is transported to the cell surface although its transmembrane domain contains a conserved arginine residue.ResultsWe expressed various chimeric proteins and analyzed the influence of their transmembrane domain on their intracellular localization. The transmembrane domain of the HIV-1 Env protein does not cause ER retention. This is not due to the presence of conserved glycine residues, or to the position of the arginine residue, but to the length of the transmembrane domain. A shortened version of the Env transmembrane domain causes arginine-dependent ER targeting. Remarkably, the transmembrane domain of the HIV-1 Env protein, although it does not confer ER retention, interacts efficiently with negatively charged residues in the membrane.ConclusionThese results suggest that the intrinsic properties of the HIV-1 Env transmembrane domain allow the protein to escape ER-retention mechanisms, while maintaining its ability to interact with cellular proteins and to influence cellular physiology.