Phosphoinositides,ezrin/moesin,and rac1 regulate fusion of rhodopsin transport carriers in retinal photoreceptors

The post-Golgi trafficking of rhodopsin in photoreceptor cells is mediated by rhodopsin-bearing transport carriers (RTCs) and regulated by the small GTPase rab8. In this work, we took a combined pharmacological-proteomic approach to uncover new regulators of RTC trafficking toward the specialized light-sensitive organelle, the rod outer segment (ROS). We perturbed phospholipid synthesis by activating phospholipase D with sphingosine 1-phosphate (S1P) or inhibiting phosphatidic acid phosphohydrolase by propranolol (Ppl). S1P stimulated the overall rate of membrane trafficking toward the ROS. Ppl stimulated budding of RTCs, but blocked membrane delivery to the ROS. Ppl caused accumulation of RTCs in the vicinity of the fusion sites, suggesting a defect in tethering, similar to the previously described phenotype of the rab8T22N mutant. Proteomic analysis of RTCs accumulated upon Ppl treatment showed a significant decrease in phosphatidylinositol-4,5-bisphosphate-binding proteins ezrin and/or moesin. Ppl induced redistribution of moesin, actin and the small GTPase rac1 from RTCs into the cytosol. By confocal microscopy, ezrin/moesin and rac1 colocalized with rab8 on RTCs at the sites of their fusion with the plasma membrane; however, this distribution was lost upon Ppl treatment. Our data suggest that in photoreceptors phosphatidylinositol-4,5-bisphosphate, moesin, actin, and rac1 act in concert with rab8 to regulate tethering and fusion of RTCs. Consequentially, they are necessary for rhodopsin-laden membrane delivery to the ROS, thus controlling the critical steps in the biogenesis of the light-detecting organelle.     

Inhibition of InhA activity,but not KasA activity,induces formation of a KasA-containing complex in mycobacteria

Isoniazid (INH) remains one of the key drugs used to control tuberculosis, with the enoyl-AcpM reductase InhA being the primary target. However, based on the observation that INH-treated Mycobacterium tuberculosis overproduces KasA, an enzyme involved in the biosynthesis of mycolic acids, and induces the formation of a covalent complex consisting of AcpM, KasA, and INH, it has been proposed that KasA represents the primary target of INH. However, the relevance of this complex to INH action remains obscure. This study was aimed at clarifying the role of InhA and KasA in relation to INH activity. By using anti-KasA antibodies we detected the KasA-containing complex in INH-treated Mycobacterium smegmatis. In addition, INH-treated cells also produced constant levels of KasA that were not sequestered in the complex and presumably were sufficient to ensure mycolic acid biosynthesis. Interestingly, a furA-lacking strain induced the complex at lower concentrations of INH compared with the control strain, whereas higher INH concentrations were necessary to induce the complex in a strain that lacks katG, suggesting that INH needs to be activated by KatG to induce the KasA-containing complex. The InhA inhibitors ethionamide and diazaborine also induced the complex; thus, its formation was not specifically relevant to INH action but was because of InhA inhibition. In addition, in vitro assays using purified InhA and KasA demonstrated that KatG-activated INH, triclosan, and diazaborine inhibited InhA but not KasA activity. Moreover, several thermosensitive InhA mutant strains of M. smegmatis constitutively expressed the KasA-containing complex. This study provides the biochemical and genetic evidence. 1) Only inhibition of InhA, but not KasA, induces the KasA-containing complex. 2) INH is not part of the complex. 3) INH does not target KasA, consistent with InhA being the primary target of INH.     

An essential role of Sam50 in the protein sorting and assembly machinery of the mitochondrial outer membrane

The preprotein translocase of the outer mitochondrial membrane (TOM complex) contains one essential subunit, the channel Tom40. The assembly pathway of the precursor of Tom40 involves the TOM complex and the sorting and assembly machinery (SAM complex) with the non-essential subunit Mas37. We have identified Sam50, the second essential protein of the mitochondrial outer membrane. Sam50 contains a beta-barrel domain conserved from bacteria to man and is a subunit of the SAM complex. Yeast mutants of Sam50 are defective in the assembly pathways of Tom40 and the abundant outer membrane protein porin, while the import of matrix proteins is not affected. Thus the protein sorting and assembly machinery of the mitochondrial outer membrane involves an essential, conserved protein.     

Some multiple-time-scale problems in molecular dynamics

Many physical problems, particularly in chemical and biological systems, involve processes that occur over widely varying time scales. Such problems have motivated the development of new methods for treating multiple-time-scale problems in molecular dynamics (MD). Methods have been developed for determining the vibrational frequencies and normal modes of large systems in full and reduced conformational space. A method is given for quasiharmonic analysis and reduced quasiharmonic analysis.     

Determination of selenium in serum by FI-HG-AAS and calculation of dietary intake

A method was developed for the determination of selenium concentration in serum by flow injection-hydride generation-atomic absorption spectrometry (FI-HG-AAS) following microwave digestion of serum samples and reduction of selenate to selenite. The detection limit of the method was 0.3 μg Se/L and the characteristic concentration, corresponding to the 0.0044 absorbance signal, was 0.12 μg Se/L. The results from the analysis of two Seronorm standard reference materials showed good agreement with the certified values. The method was then used to analyze selenium in sera of Austrian and Slovenian people for the calculation of dietary intakes. The selenium concentrations in sera of mothers at delivery, their neonates, and the male and female adults were 71 ± 14, 42 ± 6, 75 ± 21, and 65 ± 16 μg/L for the Austrians and 62 ± 15, 34 ± 7, 70 ± 12, and 66 ± 15 μg/L for the Slovenians. The dietary intakes of selenium of the mothers and the male and the female adults were calculated as 52, 37, and 46 μg/d for the Austrians and 45, 38, and 32 μg/d for the Slovenians.     

The morphology proteins Mdm12/Mmm1 function in the major beta-barrel assembly pathway of mitochondria

The beta-barrel proteins of mitochondria are synthesized on cytosolic ribosomes. The proteins are imported by the translocase of the outer membrane (TOM) and the sorting and assembly machinery (SAM). It has been assumed that the SAM(core) complex with the subunits Sam35, Sam37 and Sam50 represents the last import stage common to all beta-barrel proteins, followed by splitting in a Tom40-specific route and a route for other beta-barrel proteins. We have identified new components of the beta-barrel assembly machinery and show that the major beta-barrel pathway extends beyond SAM(core). Mdm12/Mmm1 function after SAM(core) yet before splitting of the major pathway. Mdm12/Mmm1 have been known for their role in maintenance of mitochondrial morphology but we reveal assembly of beta-barrel proteins as their primary function. Moreover, Mdm10, which functions in the Tom40-specific route, can associate with SAM(core) as well as Mdm12/Mmm1 to form distinct assembly complexes, indicating a dynamic exchange between the machineries governing mitochondrial beta-barrel assembly. We conclude that assembly of mitochondrial beta-barrel proteins represents a major function of the morphology proteins Mdm12/Mmm1.     

Autophagosomes and lipid droplets: no longer just chewing the fat

Autophagosomes are organelles capable of sequestering and degrading diverse cytoplasmic cargo for nutritional and quality control purposes. Targeted are also lipid droplets (LDs), the cytoplasmic stores of neutral lipids. In this issue of The EMBO Journal, Shpilka et al ( 2015 ) show that the relationship between LDs and autophagosomes is far more intricate and that LDs regulate autophagosome biogenesis.     

Ay, there's the Rab: organelle maturation by Rab conversion

Is endocytosed cargo transported by vesicular intermediates between early and late endosomes, or does an endosome mature while its cargo remains in place? Current work suggests that the latter takes place within the endosomal system via a process termed Rab conversion.