Eukaryotic cells can use the autophagy pathway to defend against microbes that gain access to the cytosol or reside in pathogen-modified vacuoles. It remains unclear if pathogens have evolved specific mechanisms to manipulate autophagy. Here we find that the intracellular pathogen Legionella pneumophila could interfere with autophagy using the bacterial effector protein RavZ to directly uncouple Atg8 proteins attached to phosphatidylethanolamine on autophagosome membranes. RavZ hydrolyzed the amide bond between the carboxyl-terminal glycine residue and an adjacent aromatic residue in Atg8 proteins, producing an Atg8 protein that could not be reconjugated by Atg7 and Atg3. Thus, intracellular pathogens can inhibit autophagy by irreversibly inactivating Atg8 proteins during infection.

Estimates based on proteomic analyses indicate that a third of translated proteins in eukaryotic genomes enter the secretory pathway. After folding and assembly of nascent secretory proteins in the endoplasmic reticulum (ER), the coat protein complex II (COPII) selects folded cargo for export in membrane-bound vesicles. To accommodate the great diversity in secretory cargo, protein sorting receptors are required in a number of instances for efficient ER export. These transmembrane sorting receptors couple specific secretory cargo to COPII through interactions with both cargo and coat subunits. After incorporation into COPII transport vesicles, protein sorting receptors release bound cargo in pre-Golgi or Golgi compartments, and receptors are then recycled back to the ER for additional rounds of cargo export. Distinct types of protein sorting receptors that recognize carbohydrate and/or polypeptide signals in secretory cargo have been characterized. Our current understanding of the molecular mechanisms underlying cargo receptor function are described.

The protein biochemistry supporting autophagosome growth on the cup-like isolation membrane is likely different from the biochemistry on the closed and maturing autophagosome. Thus, the highly curved rim of the cup may serve as a functionally-required surface for transiently-associated components of the early-acting autophagic machinery. Here we demonstrate that the E2-like enzyme, Atg3, facilitates LC3/GABARAPlipidation only on membranes exhibiting local lipid-packing defects. This activity requires an amino-terminal amphipathic helix similar to motifs found on proteins targeting highly curved intracellular membranes. By tuning the hydrophobicity of this motif, we can promote or inhibit lipidation in vitro and in rescue experiments in Atg3 knockout cells, implying a physiologic role for this stress detection. The need for extensive lipid-packing defects suggests that Atg3 is designed to work at highly-curved membranes perhaps including the limiting edge of the growing phagophore.

The underlying causes of type I congenital disorders of glycosylation (CDG I) have been shown to be mutations in genes encoding proteins involved in the biosynthesis of the dolichyl-linked oligosaccharide (Glc 3 Man 9 GlcNAc 2 -PP-dolichyl) that is required for protein glycosylation. Here we describe a CDG I patient displaying gastrointestinal problems but no central nervous system deficits. Fibroblasts from this patient accumulate mainly Man 9 GlcNAc 2 -PP-dolichyl, but in the presence of castanospermine, an endoplasmic reticulum glucosidase inhibitor Glc 1 Man 9 GlcNAc 2 -PP-dolichyl predominates, suggesting inefficient addition of the second glucose residue onto lipid-linked oligosaccharide. Northern blot analysis revealed the cells from the patient to possess only 10 -20% normal amounts of mRNA encoding the enzyme, dolichyl-P-glucose:Glc 1 Man 9 GlcNAc 2 -PP-dolichyl ␣3-glucosyltransferase (hALG8p), which catalyzes this reaction. Sequencing of hALG8 genomic DNA revealed exon 4 to contain a base deletion in one allele and a base insertion in the other. Both mutations give rise to premature stop codons predicted to generate severely truncated proteins, but because the translation inhibitor emetine was shown to stabilize the hALG8 mRNA from the patient to normal levels, it is likely that both transcripts undergo nonsensemediated mRNA decay. As the cells from the patient were successfully complemented with wild type hALG8 cDNA, we conclude that these mutations are the underlying cause of this new CDG I subtype that we propose be called CDG Ih.

Type I congenital disorders of glycosylation (CDG I) are diseases presenting multisystemic lesions including central and peripheral nervous system deficits. The disease is characterized by under-glycosylated serum glycoproteins and is caused by mutations in genes encoding proteins involved in the stepwise assembly of dolichol-oligosaccharide used for protein N-glycosylation. We report that fibroblasts from a type I CDG patient, born of consanguineous parents, are deficient in their capacity to add the eighth mannose residue onto the lipid-linked oligosaccharide precursor. We have characterized cDNA corresponding to the human ortholog of the yeast gene ALG12 that encodes the dolichyl-P-Man:Man 7 GlcNAc 2 -PP-dolichyl ␣6-mannosyltransferase that is thought to accomplish this reaction, and we show that the patient is homozygous for a point mutation (T571G) that causes an amino acid substitution (F142V) in a conserved region of the protein. As the pathological phenotype of the fibroblasts of the patient was largely normalized upon transduction with the wild type gene, we demonstrate that the F142V substitution is the underlying cause of this new CDG, which we suggest be called CDG Ig. Finally, we show that the fibroblasts of the patient are capable of the direct transfer of Man 7 GlcNAc 2 from dolichol onto protein and that this N-linked structure can be glucosylated by UDP-glucose: glycoprotein glucosyltransferase in the endoplasmic reticulum.

No abstract

Congenital disorders of glycosylation (CDG) type I (CDG I) are rare autosomal recessive diseases caused by deficiencies in the assembly of the dolichol-linked oligosaccharide (DLO) that is required for N-glycoprotein biosynthesis. CDG Ie is due to a defect in the synthesis of dolichyl-phosphoryl-mannose (Dol-P-Man), which is needed for DLO biosynthesis as well as for other glycosylation pathways. Human Dol-P-Man synthase is a heterotrimeric complex composed of DPM1p, DPM2p, and DPM3p, with DPM1p being the catalytic subunit. Here, we report two new CDG Ie patients who present milder symptoms than the five other CDG Ie patients described to date. The clinical pictures of the patients MS and his sister MT are dominated by major ataxia, with no notable hepatic involvement. MS cells accumulate the immature DLO species Dol-PP-GlcNAc 2 Man 5 and possess only residual Dol-P-Man synthase activity. One homozygous intronic mutation, g.IVS4 -5TϾA, was found in the DPM1 gene, leading to exon skipping and transcription of a shortened transcript. Moreover, DPM1 expression was reduced by more than 90% in MS cells, in a nonsense-mediated mRNA decay (NMD)-independent manner. Full analysis of the DPM2 and DPM3 genes revealed a decrease in DPM2 expression and normal expression of DPM3. This description emphasizes the large spectrum of symptoms characterizing CDG I patients. N-glycan biosynthesis starts by the construction of a DLO in the endoplasmic reticulum (ER) membrane. Once completed, the tetradecasaccharide (GlcNAc 2 Man 9 Glc 3 ) of the mature DLO is transferred onto nascent glycoproteins containing the consensus sequence Asn-X-Thr/Ser (where X can be any amino acid except Pro). This oligosaccharide is then trimmed by ER and Golgi-resident glycosidases and glycosyltransferases to permit the proper localization and activity of the N-glycoprotein.The last four mannose residues of the mature DLO are added by enzymes that use Dol-P-Man as a donor substrate. In mammals, Dol-P-Man synthase is composed of three subunits: DPM1p, DPM2p, and DPM3p (2). DPM2p and DPM3p are ER-resident, integral membrane proteins that interact with soluble DPM1p to ensure its correct localization and enzymic activity. Dol-P-Man is an important compound because, besides N-glycosylation, it is a substrate for O-mannosylation, glycophosphatidyl inositol (GPI)-anchor biosynthesis (3) and C-mannosylation (4). CDG I are rare autosomal recessive diseases caused by deficiencies of DLO biosynthetic enzymes. Up to 12 subtypes (CDG Ia to IL) have been described to date, each one involving a different enzymic deficiency in the pathway (5). CDG I patients show a very broad range of clinical and biologic presentations, but severe neurologic symptoms are often predominant. The variation in the symptoms of this newly discovered group of diseases makes genotype to phenotype associations difficult. Therefore, patient descriptions coupled with mutation analyses are required to get more insights into the pathophysiology of the disease. CDG Ie, which was first des...

The phagophore membrane is highly curved along the rim of the open cup, suggesting that the molecular mechanisms governing its formation and growth could rely on membrane curvature-dependent events. To this end, we recently reported that lipidation of the LC3 protein family is facilitated on highly curved membranes in vitro. We further showed that the conjugating enzyme ATG3 contains an amphipathic helix that is responsible for this membrane curvature dependency, and that the maintenance of this amphipathic structure is essential for ATG3 function in vivo.