The organization of secretory traffic remains unclear, mainly because of the complex structure and dynamics of the secretory pathway. We have thus studied a simplified system, a single synchronized traffic wave crossing an individual Golgi stack, using electron tomography. Endoplasmic-reticulum-to-Golgi carriers join the stack by fusing with cis cisternae and induce the formation of intercisternal tubules, through which they redistribute their contents throughout the stack. These tubules seem to be pervious to Golgi enzymes, whereas Golgi vesicles are depleted of both enzymes and cargo. Cargo then traverses the stack without leaving the cisternal lumen. When cargo exits the stack, intercisternal connections disappear. These findings provide a new view of secretory traffic that includes dynamic intercompartment continuities as key players.

No abstract

The enzyme phospholipase A2 (cPLA2α) is involved in the formation of intercisternal tubules that mediate transport of proteins within the Golgi complex.

The delivery of mannose 6-phosphate receptors carrying lysosomal hydrolases from the trans-Golgi network (TGN) to the endosomal system is mediated by selective incorporation of the receptor-hydrolase complexes into vesicular transport carriers (TCs) that are coated with clathrin and the adaptor proteins, GGA and AP-1. Previous electron microscopy (EM) and biochemical studies have shown that these TCs consist of spherical coated vesicles with a diameter of 60-100 nm. The use of fluorescent live cell imaging, however, has revealed that at least some of this transport relies on a subset of apparently larger and highly pleiomorphic carriers that detach from the TGN and translocate toward the peripheral cytoplasm until they meet with distally located endosomes. The ultrastructure of such long-range TCs has remained obscure because of the inability to examine by conventional EM the morphological details of rapidly moving organelles. The recent development of correlative light-EM has now allowed us to obtain ultrastructural 'snapshots' of these TCs immediately after their formation from the TGN in live cells. This approach has revealed that such carriers range from typical 60-to 100-nm clathrin-coated vesicles to larger, convoluted tubularvesicular structures displaying several coated buds. We propose that this subset of TCs serve as vehicles for longrange distribution of biosynthetic or recycling cargo from the TGN to the peripheral endosomes.

Acylated SH4 domains represent N-terminal targeting signals that anchor peripheral membrane proteins such as Src kinases in the inner leaflet of plasma membranes.Here we provide evidence for a novel regulatory mechanism that may control the levels of SH4 proteins being associated with plasma membranes. Using a fusion protein of the SH4 domain of Leishmania HASPB and GFP as a model system, we demonstrate that threonine 6 is a substrate for phosphorylation. Substitution of threonine 6 by glutamate (to mimic a phosphothreonine residue) resulted in a dramatic redistribution from plasma membranes to intracellular sites with a particular accumulation in a perinuclear region. As shown by both pharmacological inhibition and RNAi-mediated downregulation of the threonine/ serine-specific phosphatases PP1 and PP2A, recycling back to the plasma membrane required dephosphorylation of threonine 6. We provide evidence that a cycle of phosphorylation and dephosphorylation may also be involved in intracellular targeting of other SH4 proteins such as the Src kinase Yes.