Isolation of an intercisternal matrix that binds medial-Golgi enzymes

Purified rat-liver Golgi stacks were extracted in a buffer containing 2% (w/v) TX-100, 50mM MOPS pH7.0, 0.1mM MgCl2, 1mM DTT and 10% (w/v) sucrose, and centrifuged to produce an insoluble pellet which contained the majority of three medial-Golgi enzymes, mannosidase II and N-acetylglucosaminyltransferases I and II. Proteins from other regions of the Golgi stack were mostly solubilised. A further extraction of this pellet in the TX-100 buffer containing 150mM NaCl led to complete solubilisation of these medial-Golgi enzymes. After the salt extraction, a second insoluble pellet was produced which was termed the Golgi matrix. The salt-solubilised medial-enzymes could rebind the matrix upon dialysis, while an enzyme from the trans-Golgi could not. Scatchard analysis revealed that rebinding was saturable and occurred with a high affinity, suggesting that the matrix contained a fixed number of receptors which specifically bound the medial-enzymes. This suggested that the enzyme insolubility in detergent was due to their interaction with the matrix. Digestion of intact Golgi membranes with proteinase K greatly increased the detergent-solubility to the medial-enzymes, suggesting that components of the matrix were present on the cytoplasmic, intercistemal face of the Golgi membranes. This topological orientation suggested that the matrix might play a role in stacking the Golgi cisternae. Binding of the enzymes did not, however, occur via their cytoplasmic or membrane-spanning domains, suggesting that the matrix is a complex structure, containing components on both sides of the Golgi membrane. Because of its topology and its binding capacity for medial-enzymes, the matrix may function in aiding the retention of Golgi proteins, maintaining the flattened cisternal morphology or in connecting adjacent cisternae to form the characteristic Golgi stack

I2B is a Small Cytosolic Protein that Participates in Vacuole Fusion

Saccharomyces cerevisiae vacuole inheritance requires two low molecular weight activities, LMA1 and LMA2. LMA1 is a heterodimer of thioredoxin and protease B inhibitor 2 (IB2). Here we show that the second low molecular weight activity (LMA2) is monomeric IB2. Though LMA2 / IB2 was initially identified as a protease B inhibitor, this protease inhibitor activity is not related to its ability to promote vacuole fusion: ( i ) Low M r protease B inhibitors cannot substitute for LMA1 or LMA2, ( ii ) LMA1 and LMA2 promote the fusionof vacuoles from a strain that has no protease B, ( iii ) low concentrations of LMA2 that fully inhibit protease B do not promote vacuole fusion, and ( iv ) LMA1, in which is complexed with thioredoxin,is far more active than LMA2 / IB2 in promoting vacuole fusion and far less active in inhibiting protease B. These studies establish a new function for IB2

A Vacuolar v–t-SNARE Complex, the Predominant Form In Vivo and on Isolated Vacuoles, Is Disassembled and Activated for Docking and Fusion

Homotypic vacuole fusion in yeast requires Sec18p (N-ethylmaleimide–sensitive fusion protein [NSF]), Sec17p (soluble NSF attachment protein [α-SNAP]), and typical vesicle (v) and target membrane (t) SNAP receptors (SNAREs). We now report that vacuolar v- and t-SNAREs are mainly found with Sec17p as v–t-SNARE complexes in vivo and on purified vacuoles rather than only transiently forming such complexes during docking, and disrupting them upon fusion. In the priming reaction, Sec18p and ATP dissociate this v–t-SNARE complex, accompanied by the release of Sec17p. SNARE complex structure governs each functional aspect of priming, as the v-SNARE regulates the rate of Sec17p release and, in turn, Sec17p-dependent SNARE complex disassembly is required for independent function of the two SNAREs. Sec17p physically and functionally interacts largely with the t-SNARE. (a) Antibodies to the t-SNARE, but not the v-SNARE, block Sec17p release. (b) Sec17p is associated with the t-SNARE in the absence of v-SNARE, but is not bound to the v-SNARE without t-SNARE. (c) Vacuoles with t-SNARE but no v-SNARE still require Sec17p/Sec18p priming, whereas their fusion partners with v-SNARE but no t-SNARE do not. Sec18p thus acts, upon ATP hydrolysis, to disassemble the v–t-SNARE complex, prime the t-SNARE, and release the Sec17p to allow SNARE participation in docking and fusion. These studies suggest that the analogous ATP-dependent disassembly of the 20-S complex of NSF, α-SNAP, and v- and t-SNAREs, which has been studied in detergent extracts, corresponds to the priming of SNAREs for docking rather than to the fusion of docked membranes

Role of NAD+ and ADP-Ribosylation in the Maintenance of the Golgi Structure

We have investigated the role of the ADP- ribosylation induced by brefeldin A (BFA) in the mechanisms controlling the architecture of the Golgi complex. BFA causes the rapid disassembly of this organelle into a network of tubules, prevents the association of coatomer and other proteins to Golgi membranes, and stimulates the ADP-ribosylation of two cytosolic proteins of 38 and 50 kD (GAPDH and BARS-50; De Matteis, M.A., M. DiGirolamo, A. Colanzi, M. Pallas, G. Di Tullio, L.J. McDonald, J. Moss, G. Santini, S. Bannykh, D. Corda, and A. Luini. 1994. Proc. Natl. Acad. Sci. USA. 91:1114–1118; Di Girolamo, M., M.G. Silletta, M.A. De Matteis, A. Braca, A. Colanzi, D. Pawlak, M.M. Rasenick, A. Luini, and D. Corda. 1995. Proc. Natl. Acad. Sci. USA. 92:7065–7069). To study the role of ADP-ribosylation, this reaction was inhibited by depletion of NAD+ (the ADP-ribose donor) or by using selective pharmacological blockers in permeabilized cells. In NAD+-depleted cells and in the presence of dialized cytosol, BFA detached coat proteins from Golgi membranes with normal potency but failed to alter the organelle's structure. Readdition of NAD+ triggered Golgi disassembly by BFA. This effect of NAD+ was mimicked by the use of pre–ADP- ribosylated cytosol. The further addition of extracts enriched in native BARS-50 abolished the ability of ADP-ribosylated cytosol to support the effect of BFA. Pharmacological blockers of the BFA-dependent ADP-ribosylation (Weigert, R., A. Colanzi, A. Mironov, R. Buccione, C. Cericola, M.G. Sciulli, G. Santini, S. Flati, A. Fusella, J. Donaldson, M. DiGirolamo, D. Corda, M.A. De Matteis, and A. Luini. 1997. J. Biol. Chem. 272:14200–14207) prevented Golgi disassembly by BFA in permeabilized cells. These inhibitors became inactive in the presence of pre–ADP-ribosylated cytosol, and their activity was rescued by supplementing the cytosol with a native BARS-50–enriched fraction. These results indicate that ADP-ribosylation plays a role in the Golgi disassembling activity of BFA, and suggest that the ADP-ribosylated substrates are components of the machinery controlling the structure of the Golgi apparatus

Rab6 and Rab11 Regulate Chlamydia trachomatis Development and Golgin-84-Dependent Golgi Fragmentation

Many intracellular pathogens that replicate in special membrane bound compartments exploit cellular trafficking pathways by targeting small GTPases, including Rab proteins. Members of the Chlamydiaceae recruit a subset of Rab proteins to their inclusions, but the significance of these interactions is uncertain. Using RNA interference, we identified Rab6 and Rab11 as important regulators of Chlamydia infections. Depletion of either Rab6 or Rab11, but not the other Rab proteins tested, decreased the formation of infectious particles. We further examined the interplay between these Rab proteins and the Golgi matrix components golgin-84 and p115 with regard to Chlamydia-induced Golgi fragmentation. Silencing of the Rab proteins blocked Chlamydia-induced and golgin-84 knockdown-stimulated Golgi disruption, whereas Golgi fragmentation was unaffected in p115 depleted cells. Interestingly, p115-induced Golgi fragmentation could rescue Chlamydia propagation in Rab6 and Rab11 knockdown cells. Furthermore, transport of nutrients to Chlamydia, as monitored by BODIPY-Ceramide, was inhibited by Rab6 and Rab11 knockdown. Taken together, our results demonstrate that Rab6 and Rab11 are key regulators of Golgi stability and further support the notion that Chlamydia subverts Golgi structure to enhance its intracellular development