Blocking variant surface glycoprotein synthesis alters endoplasmic reticulum exit sites/Golgi homeostasis in Trypanosoma brucei

The predominant secretory cargo of bloodstream form Trypanosoma brucei is Variant Surface Glycoprotein (VSG), comprising ~10% total protein and forming a dense protective layer. Blocking VSG translation using Morpholino oligonucleotides triggered a precise pre/cytokinesis arrest. We investigated the effect of blocking VSG synthesis on the secretory pathway. The number of Golgi decreased, particularly in post/mitotic cells, from 3.5 ± 0.6 to 2.0 ± 0.04 per cell. Similarly, the number of ER exit sites (ERES) in post/mitotic cells dropped from (3.9 ± 0.6) to (2.7 ± 0.1) eight hours after blocking VSG synthesis. The secretory pathway was still functional in these stalled cells, as monitored using Cathepsin L. Rates of phospholipid and GPI/anchor biosynthesis were relatively unaffected, except for the level of sphingomyelin which increased. However, both ER and Golgi morphology became distorted, with the Golgi cisternae becoming significantly dilated, particularly at the trans/face. Membrane accumulation in these structures is possibly caused by reduced budding of nascent vesicles due to the drastic reduction in the total amount of secretory cargo, i.e. VSG. These data argue that the total flux of secretory cargo impacts upon the biogenesis and maintenance of secretory structures and organelles in T. brucei including the ERES and Golgi

Asymmetric Requirements for a Rab Gtpase and Snare Proteins in Fusion of Copii Vesicles with Acceptor Membranes

Soluble NSF attachment protein receptor (SNARE) proteins are essential for membrane fusion in transport between the yeast ER and Golgi compartments. Subcellular fractionation experiments demonstrate that the ER/Golgi SNAREs Bos1p, Sec22p, Bet1p, Sed5p, and the Rab protein, Ypt1p, are distributed similarly but localize primarily with Golgi membranes. All of these SNARE proteins are efficiently packaged into COPII vesicles and suggest a dynamic cycling of SNARE machinery between ER and Golgi compartments. Ypt1p is not efficiently packaged into vesicles under these conditions. To determine in which membranes protein function is required, temperature-sensitive alleles of BOS1, BET1, SED5, SLY1, and YPT1 that prevent ER/Golgi transport in vitro at restrictive temperatures were used to selectively inactivate these gene products on vesicles or on Golgi membranes. Vesicles bearing mutations in Bet1p or Bos1p inhibit fusion with wild-type acceptor membranes, but acceptor membranes containing these mutations are fully functional. In contrast, vesicles bearing mutations in Sed5p, Sly1p, or Ypt1p are functional, whereas acceptor membranes containing these mutations block fusion. Thus, this set of SNARE proteins is symmetrically distributed between vesicle and acceptor compartments, but they function asymmetrically such that Bet1p and Bos1p are required on vesicles and Sed5p activity is required on acceptor membranes. We propose the asymmetry in SNARE protein function is maintained by an asymmetric distribution and requirement for the Ypt1p GTPase in this fusion event. When a transmembrane-anchored form of Ypt1p is used to restrict this GTPase to the acceptor compartment, vesicles depleted of Ypt1p remain competent for fusion