A genome-wide RNA interference screen identifies two novel components of the metazoan secretory pathway

Genetic screens in the yeast Saccharomyces cerevisiae have identified many proteins involved in the secretory pathway, most of which have orthologues in higher eukaryotes. To investigate whether there are additional proteins that are required for secretion in metazoans but are absent from yeast, we used genome-wide RNA interference (RNAi) to look for genes required for secretion of recombinant luciferase from Drosophila S2 cells. This identified two novel components of the secretory pathway that are conserved from humans to plants. Gryzun is distantly related to, but distinct from, the Trs130 subunit of the TRAPP complex but is absent from S. cerevisiae. RNAi of human Gryzun (C4orf41) blocks Golgi exit. Kish is a small membrane protein with a previously uncharacterised orthologue in yeast. The screen also identified Drosophila orthologues of almost 60% of the yeast genes essential for secretion. Given this coverage, the small number of novel components suggests that contrary to previous indications the number of essential core components of the secretory pathway is not much greater in metazoans than in yeasts

Biallelic missense variants in COG3 cause a congenital disorder of glycosylation with impairment of retrograde vesicular trafficking

Biallelic variants in genes for seven out of eight subunits of the conserved oligomeric Golgi complex (COG) are known to cause recessive congenital disorders of glycosylation (CDG) with variable clinical manifestations. COG3 encodes a constituent subunit of the COG complex that has not been associated with disease traits in humans. Herein, we report two COG3 homozygous missense variants in four individuals from two unrelated consanguineous families that co-segregated with COG3–CDG presentations. Clinical phenotypes of affected individuals include global developmental delay, severe intellectual disability, microcephaly, epilepsy, facial dysmorphism, and variable neurological findings. Biochemical analysis of serum transferrin from one family showed the loss of a single sialic acid. Western blotting on patient-derived fibroblasts revealed reduced COG3 and COG4. Further experiments showed delayed retrograde vesicular recycling in patient cells. This report adds to the knowledge of the COG–CDG network by providing collective evidence for a COG3–CDG rare disease trait and implicating a likely pathology of the disorder as the perturbation of Golgi trafficking

Importance of the N-Terminal Domain of the Qb-SNARE Vti1p for Different Membrane Transport Steps in the Yeast Endosomal System

Gossing M, Chidambaram S, Fischer von Mollard G. Importance of the N-Terminal Domain of the Qb-SNARE Vti1p for Different Membrane Transport Steps in the Yeast Endosomal System. Plos One. 2013;8(6): e66304.SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) on transport vesicles and target membranes are crucial for vesicle targeting and fusion. They form SNARE complexes, which contain four a-helical SNARE motifs contributed by three or four different SNAREs. Most SNAREs function only in a single transport step. The yeast SNARE Vti1p participates in four distinct SNARE complexes in transport from the trans Golgi network to late endosomes, in transport to the vacuole, in retrograde transport from endosomes to the trans Golgi network and in retrograde transport within the Golgi. So far, all vti1 mutants investigated had mutations within the SNARE motif. Little is known about the function of the N-terminal domain of Vti1p, which forms a three helix bundle called H-abc domain. Here we generated a temperature-sensitive mutant of this domain to study the effects on different transport steps. The secondary structure of wild type and vti1-3 H-abc domain was analyzed by circular dichroism spectroscopy. The amino acid exchanges identified in the temperature-sensitive vti1-3 mutant caused unfolding of the H-abc domain. Transport pathways were investigated by immunoprecipitation of newly synthesized proteins after pulse-chase labeling and by fluorescence microscopy of a GFP-tagged protein cycling between plasma membrane, early endosomes and Golgi. In vti1-3 cells transport to the late endosome and assembly of the late endosomal SNARE complex was blocked at 37 degrees C. Retrograde transport to the trans Golgi network was affected while fusion with the vacuole was possible but slower. Steady state levels of SNARE complexes mediating these steps were less affected than that of the late endosomal SNARE complex. As different transport steps were affected our data demonstrate the importance of a folded Vti1p H-abc domain for transport