The Dsl1 protein tethering complex is a resident endoplasmic reticulum complex, which interacts with five soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptors (SNAREs): implications for fusion and fusion regulation.

Retrograde vesicular transport from the Golgi to the ER requires the Dsl1 tethering complex, which consists of the three subunits Dsl1, Dsl3, and Tip20. It forms a stable complex with the SNAREs Ufe1, Use1, and Sec20 to mediate fusion of COPI vesicles with the endoplasmic reticulum. Here, we analyze molecular interactions between five SNAREs of the ER (Ufe1, Use1, Sec20, Sec22, and Ykt6) and the Dsl1 complex in vitro and in vivo. Of the two R-SNAREs, Sec22 is preferred over Ykt6 in the Dsl-SNARE complex. The NSF homolog Sec18 can displace Ykt6 but not Sec22, suggesting a regulatory function for Ykt6. In addition, our data also reveal that subunits of the Dsl1 complex (Dsl1, Dsl3, and Tip20), as well as the SNAREs Ufe1 and Sec20, are ER-resident proteins that do not seem to move into COPII vesicles. Our data support a model, in which a tethering complex is stabilized at the organelle membrane by binding to SNAREs, recognizes the incoming vesicle via its coat and then promotes its SNARE-mediated fusion.

Analysis of DHHC acyltransferases implies overlapping substrate specificity and a two-step reaction mechanism.

Asp-His-His-Cys (DHHC) cysteine-rich domain (CRD) acyltransferases are polytopic transmembrane proteins that are found along the endomembrane system of eukaryotic cells and mediate palmitoylation of peripheral and integral membrane proteins. Here, we address the in vivo substrate specificity of five of the seven DHHC acyltransferases for peripheral membrane proteins by an overexpression approach. For all analysed DHHC proteins we detect strongly overlapping substrate specificity. In addition, we now show acyltransferase activity for Pfa5. More importantly, the DHHC protein Pfa3 is able to trap several substrates at the vacuole. For Pfa3 and its substrate Vac8, we can distinguish two consecutive steps in the acylation reaction: an initial binding that occurs independently of its central cysteine in the DHHC box, but requires myristoylation of its substrate Vac8, and a DHHC-motif dependent acylation. Our data also suggest that proteins can be palmitoylated on several organelles. Thus, the intracellular distribution of DHHC proteins provides an acyltransferase network, which may promote dynamic membrane association of substrate proteins.

Depalmitoylation of Ykt6 prevents its entry into the multivesicular body pathway.

The dually lipidated SNARE Ykt6 is found on intracellular membranes and in the cytosol. In this study, we show that Ykt6 localizes to the Golgi as well as endosomal and vacuolar membranes in vivo. The ability of Ykt6 to cycle between the cytosol and the membranes depends on the intramolecular interaction of the N-terminal longin and C-terminal SNARE domains and not on either domain alone. A mutant deficient in this interaction accumulates on membranes and--in contrast to the wild-type protein--does not get released from vacuoles. Our data also indicate that Ykt6 is a substrate of the DHHC (Asp-His-His-Cys) acyltransferase network. Overexpression of the vacuolar acyltransferase Pfa3 drives the F42S mutant not only to the vacuole but also into the vacuolar lumen. Thus, depalmitoylation and release of Ykt6 are needed for its recycling and to circumvent its entry into the endosomal multivesicular body pathway.

The neuropeptide PACAP promotes the alpha-secretase pathway for processing the Alzheimer amyloid precursor protein.

The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) has neurotrophic as well as anti-apoptotic properties and is involved in learning and memory processes. Its specific G protein-coupled receptor PAC1 is expressed in several central nervous system (CNS) regions, including the hippocampal formation. Here we examined the effect of PAC1 receptor activation on alpha-secretase cleavage of the amyloid precursor protein (APP) and the production of secreted APP (APPsalpha). Stimulation of endogenously expressed PAC1 receptors with PACAP in human neuroblastoma cells increased APPsalpha secretion, which was completely inhibited by the PAC1 receptor specific antagonist PACAP-(6-38). In HEK cells stably overexpressing functional PAC1 receptors, PACAP-27 and PACAP-38 strongly stimulated alpha-secretase cleavage of APP. The PACAP-induced APPsalpha production was dose dependent and saturable. This increase of alpha-secretase activity was completely abolished by hydroxamate-based metalloproteinase inhibitors, including a preferential ADAM 10 inhibitor. By using several specific protein kinase inhibitors, we show that the MAP-kinase pathway [including extracellular-regulated kinase (ERK) 1 and ERK2] and phosphatidylinositol 3-kinase mediate the PACAP-induced alpha-secretase activation. Our findings provide evidence for a role of the neuropeptide PACAP in stimulation of the nonamyloidogenic pathway, which might be related to its neuroprotective properties.

Effects of copper on CHO cells: insights from gene expression analyses.

Copper concentration can impact lactate metabolism in Chinese Hamster ovary (CHO) cells. In our previous study, a 20-fold increase in initial copper concentration enabled CHO cultures to shift from net lactate production to net lactate consumption, and achieve higher cell growth and productivity. In this follow-up study, we used transcriptomics to investigate the mechanism of action (MOA) of copper that mediates this beneficial metabolism shift. From microarray profiling (days 0-7), the number of differentially expressed genes increased considerably after the lactate shift (>day 3). To uncouple the effects of copper at early time points (days 0-3) from that of lactate per se (>day 3), and to validate microarray hits, we analyzed samples before the lactate shift by RNA-Seq. Out of 6,398 overlapping genes analyzed by both transcriptomic methods, only the early growth response 1 gene-coding for a transcription factor that activates signaling pathways in response to environmental stimuli-satisfied the differential expression criteria (fold change ≥ 1.5; P < 0.05). Gene expression correlation and biological pathway analyses further confirmed that copper differences exerted minimal transcriptional impact on the CHO cultures before the lactate shift. By contrast, genes associated with hypoxia network and oxidative stress response were upregulated after the lactate shift. These upregulations should boost cell proliferation and survival, but do not account for the preceding shift in lactate metabolism. The findings here indicate that the primary MOA of copper that enabled the shift in lactate metabolism is not at the transcriptional level.

Yeast vacuole fusion: a model system for eukaryotic endomembrane dynamics.

Vesicular transport in eukaryotic cells is concluded with the consumption of the vesicle at the target membrane. This fusion process relies on Rabs, tethers and SNAREs. Powerful in vitro fusion systems using isolated organelles were crucial to obtain insights into the underlying mechanism of membrane fusion- from the initiation of fusion to lipid bilayer mixing. Among these systems, yeast vacuoles turned out to be particularly useful as they can be manipulated biochemically and genetically. Studies relying on this organelle have revealed insights into the connection of vacuole fusion to endomembrane biogenesis. A number of fusion factors were identified and characterized over the last several years, and placed into the fusion cascade. Within this review, we will present and discuss the current state of our knowledge on vacuole fusion.

Probing protein palmitoylation at the yeast vacuole.

A protein's function depends on its localization to the right cellular compartment. A number of proteins require lipidation to associate with membranes. Protein palmitoylation is a reversible lipid modification and has been shown to mediate both membrane localization and control protein function. At the yeast vacuole, several palmitoylated proteins have been identified that are required for vacuole biogenesis, including the fusion factor Vac8, the SNARE Ykt6 and the casein kinase Yck3. Moreover, both the DHHC-CRD acyltransferase Pfa3 and Ykt6 are involved in palmitoylation at the vacuole Here, we present and discuss methods to probe for protein palmitoylation at vacuoles.

Palmitoylation determines the function of Vac8 at the yeast vacuole.

Palmitoylation stably anchors specific proteins to membranes, but may also have a direct effect on the function of a protein. The yeast protein Vac8 is required for efficient vacuole fusion, inheritance and cytosol-to-vacuole trafficking. It is anchored to vacuoles by an N-terminal myristoylation site and three palmitoylation sites, also known as the SH4 domain. Here, we address the role of Vac8 palmitoylation and show that the position and number of substrate cysteines within the SH4 domain determine the vacuole localization of Vac8: stable vacuole binding of Vac8 requires two cysteines within the N-terminus, regardless of the combination. Importantly, our data suggest that palmitoylation adds functionality to Vac8 beyond simple localization. A mutant Vac8 protein, in which the palmitoylation sites were replaced by a stretch of basic residues, still localizes to vacuole membranes and functions in cytosol-to-vacuole transport, but can only complement the function of Vac8 in morphology and inheritance if it also contains a single cysteine within the SH4 domain. Our data suggest that palmitoylation is not a mere hydrophobic anchor required solely for localization, but influences the protein function(s).

ATP-independent control of Vac8 palmitoylation by a SNARE subcomplex on yeast vacuoles.

Yeast vacuole fusion requires palmitoylated Vac8. We previously showed that Vac8 acylation occurs early in the fusion reaction, is blocked by antibodies against Sec18 (yeast N-ethylmaleimide-sensitive fusion protein (NSF)), and is mediated by the R-SNARE Ykt6. Here we analyzed the regulation of this reaction on purified vacuoles. We show that Vac8 acylation is restricted to a narrow time window, is independent of ATP hydrolysis by Sec18, and is stimulated by the ion chelator EDTA. Analysis of vacuole protein complexes indicated that Ykt6 is part of a complex distinct from the second R-SNARE, Nyv1. We speculate that during vacuole fusion, Nyv1 is the classical R-SNARE, whereas the Ykt6-containing complex has a novel function in Vac8 palmitoylation.