Topological and Enzymatic Analysis of Human Alg2 Mannosyltransferase

N-glycosylation starts with the biosynthesis of lipid-linked oligosaccharide (LLO) on the endoplasmic reticulum. Alg2 mannosyltransferase adds both the α1,3- and α1,6-Man onto ManGlcNAc2-pyrophosphate-dolichol (M1Gn2-PDol) in either order to generate the branched M3Gn2-PDol product. The well-studied yeast Alg2 interacts with ER membrane through four hydrophobic domains. Unexpectedly, we show that Alg2 structure has diverged significantly between yeast and humans. Human Alg2 (hAlg2) associates with the ER via a single membrane-binding domain and is markedly more stable in vitro. These properties were exploited to develop an LC-MS quantitative kinetic assay for studying purified hAlg2. Under physiological conditions, hAlg2 prefers to transfer α1,3-Man on to M1Gn2 before adding the α1,6-Man. However, this bias is altered by an excess of GDP-Man donor or an increased level of M1Gn2 substrate, both of which trigger production of the M2Gn2 (α-1,6)-PDol. These results suggest that Alg2 may regulate the LLO biosynthetic pathway by controlling accumulation of M2Gn2 (α-1,6) intermediate.

Proteome and Lipidome of Plant Cell Membranes during Development

Intensive development of systemic biology involves intensification of such branches as proteomics and lipidomics, which are valid for systemic biology of plants. This trend is obvious due to the rapidly growing number of publications on proteomes and lipidomes of plant cells, tissues, and whole organs. Particulars of the plant nuclei, mitochondria, and chloroplasts have been rather well detailed in this regard. However, these data are scarce concerning the tonoplast, Golgi apparatus, endoplasmic reticulum, and other single-membrane organelles of the plant cell. This review surveys the current concepts related to specificity of protein and lipid spectra in the membrane structures of plant cells. The little data describing changes in these parameters in the course of development and under stress pressure are also analyzed.

GABARAP membrane conjugation sequesters the FLCN-FNIP tumor suppressor complex to activate TFEB and lysosomal biogenesis

Adaptive changes in lysosomal capacity are driven by the transcription factors TFEB and TFE3 in response to increased autophagic flux and endolysosomal stress, yet the molecular details of their activation are unclear. LC3 and GABARAP members of the ATG8 protein family are required for selective autophagy and sensing perturbation within the endolysosomal system. Here we show that during single membrane ATG8 conjugation (SMAC), Parkin-dependent mitophagy, and Salmonella-induced xenophagy, the membrane conjugation of GABARAP, but not LC3, is required for activation of TFEB/TFE3 to control lysosomal homeostasis and capacity. GABARAP directly binds to a novel LC3-interacting motif (LIR) in the FLCN/FNIP tumor suppressor complex with picomolar affinity and regulates its relocalization to these GABARAP-conjugated membrane compartments. This disrupts the regulation of RagC/D by the FLCN/FNIP GAP complex, resulting in impaired mTOR-dependent phosphorylation of TFEB without changing mTOR activity towards other substrates. Thus, the GABARAP-FLCN/FNIP-TFEB axis serves as a universal molecular sensor that coordinates lysosomal homeostasis with perturbations and cargo flux within the autophagy-lysosomal network.