Michael Veit

Vac8p release from the SNARE complex and its palmitoylation are coupled and essential for vacuole fusion

Activated fatty acids stimulate budding and fusion in several cell‐free assays for vesicular transport. This stimulation is thought to be due to protein palmitoylation, but relevant substrates have not yet been identified. We now report that Vac8p, a protein known to be required for vacuole inheritance, becomes palmitoylated when isolated yeast vacuoles are incubated under conditions that allow membrane fusion. Similar requirements for Vac8p palmitoylation and vacuole fusion, the inhibition of vacuole fusion by antibodies to Vac8p and the strongly reduced fusion of vacuoles lacking Vac8p suggest that palmitoylated Vac8p is essential for homotypic vacuole fusion. Strikingly, palmitoylation of Vac8p is blocked by the addition of antibodies to Sec18p (yeast NSF) only. Consistent with this, a portion of Vac8p is associated with the SNARE complex on vacuoles, which is lost during Sec18p‐ and ATP‐dependent priming. During or after SNARE complex disassembly, palmitoylation occurs and anchors Vac8p to the vacuolar membrane. We propose that palmitoylation of Vac8p is regulated by the same machinery that controls membrane fusion.

S Acylation of the Hemagglutinin of Influenza Viruses: Mass Spectrometry Reveals Site-Specific Attachment of Stearic Acid to a Transmembrane Cysteine

ABSTRACT S acylation of cysteines located in the transmembrane and/or cytoplasmic region of influenza virus hemagglutinins (HA) contributes to the membrane fusion and assembly of virions. Our results from using mass spectrometry (MS) show that influenza B virus HA possessing two cytoplasmic cysteines contains palmitate, whereas HA-esterase-fusion glycoprotein of influenza C virus having one transmembrane cysteine is stearoylated. HAs of influenza A virus having one transmembrane and two cytoplasmic cysteines contain both palmitate and stearate. MS analysis of recombinant viruses with deletions of individual cysteines, as well as tandem-MS sequencing, revealed the surprising result that stearate is exclusively attached to the cysteine positioned in the transmembrane region of HA.

The SNARE Ykt6 mediates protein palmitoylation during an early stage of homotypic vacuole fusion

The NSF homolog Sec18 initiates fusion of yeast vacuoles by disassembling cis‐SNARE complexes during priming. Sec18 is also required for palmitoylation of the fusion factor Vac8, although the acylation machinery has not been identified. Here we show that the SNARE Ykt6 mediates Vac8 palmitoylation and acts during a novel subreaction of vacuole fusion. This subreaction is controlled by a Sec17‐independent function of Sec18. Our data indicate that Ykt6 presents Pal‐CoA via its N‐terminal longin domain to Vac8, while transfer to Vac8s SH4 domain occurs spontaneously and not enzymatically. The conservation of Ykt6 and its localization to several organelles suggest that its acyltransferase activity may also be required in other intracellular fusion events.

FLIM-FRET and FRAP reveal association of influenza virus haemagglutinin with membrane rafts

It has been supposed that the HA (haemagglutinin) of influenza virus must be recruited to membrane rafts to perform its function in membrane fusion and virus budding. In the present study, we aimed at substantiating this association in living cells by biophysical methods. To this end, we fused the cyan fluorescent protein Cer (Cerulean) to the cytoplasmic tail of HA. Upon expression in CHO (Chinese-hamster ovary) cells HA-Cer was glycosylated and transported to the plasma membrane in a similar manner to authentic HA. We measured FLIM-FRET (Förster resonance energy transfer by fluorescence lifetime imaging microscopy) and showed strong association of HA-Cer with Myr-Pal-YFP (myristoylated and palmitoylated peptide fused to yellow fluorescent protein), an established marker for rafts of the inner leaflet of the plasma membrane. Clustering was significantly reduced when rafts were disintegrated by cholesterol extraction and when the known raft-targeting signals of HA, the palmitoylation sites and amino acids in its transmembrane region, were removed. FRAP (fluorescence recovery after photobleaching) showed that removal of raft-targeting signals moderately increased the mobility of HA in the plasma membrane, indicating that the signals influence access of HA to slowly diffusing rafts. However, Myr-Pal-YFP exhibited a much faster mobility compared with HA-Cer, demonstrating that HA and the raft marker do not diffuse together in a stable raft complex for long periods of time.

Association of Influenza Virus Proteins with Membrane Rafts

Assembly and budding of influenza virus proceeds in the viral budozone, a domain in the plasma membrane with characteristics of cholesterol/sphingolipid-rich membrane rafts. The viral transmembrane glycoproteins hemagglutinin (HA) and neuraminidase (NA) are intrinsically targeted to these domains, while M2 is seemingly targeted to the edge of the budozone. Virus assembly is orchestrated by the matrix protein M1, binding to all viral components and the membrane. Budding progresses by protein- and lipid-mediated membrane bending and particle scission probably mediated by M2. Here, we summarize the experimental evidence for this model with emphasis on the raft-targeting features of HA, NA, and M2 and review the functional importance of raft domains for viral protein transport, assembly and budding, environmental stability, and membrane fusion.

The M2 protein of influenza A virus is acylated

The M2 protein of influenza A virus, a 97 amino acid integral membrane protein expressed on the surface of infected cells, is covalently modified with long chain fatty acids. The fatty acid bond is sensitive to treatment with neutral hydroxylamine and mercaptoethanol, which indicates a labile thioester type linkage. Thin-layer chromatographic fatty acid analysis of [3H]myristic and [3H]palmitic acid-labelled M2 protein shows that palmitic acid is the predominant fatty acid linked to this polypeptide. Palmitoylation of M2 occurs post-translationally and causes an upward shift in the SDS-PAGE mobility of the protein.

The relevance of salt bridges for the stability of the influenza virus hemagglutinin

Hemagglutinin (HA) of influenza virus undergoes an irreversible conformational change at acidic pH, mediating viral fusion with the host endosomal membrane. To unravel the molecular basis of the pH‐dependent stability of HA, we demonstrate by mu‐tagenesis of the prototype HA of virus strain X31 (H3 subtype) that salt bridges, especially a tetrad salt bridge within the monomers, are crucial for folding and stability of the trimeric ectodomain. This complex (tetrad) salt bridge is highly conserved among influenza virus subtypes. Introducing additional sites of electrostatic attraction between monomers in the distal region enhanced the stability of ectodomain at low pH mimicking the natural variant H2 subtype. We propose that distinct salt bridges in the distal domain may contribute to the enhanced stability of HA of natural virus vari‐ants. This hypothesis may provide clues to understanding adaptations of virus strains (for example, avian influenza viruses) in order to preserve stability of the protein in the host‐specific environment.—Rachakonda, P. S., Veit, M., Korte, T., Ludwig, K., Böttcher, C., Huang, Q., Schmidt, M. F. G., Herrmann, A. The relevance of salt bridges for the stability of the influenza virus hemagglutinin. FASEB J. 21, 995–1002 (2007)

Synaptobrevin 2 Is Palmitoylated in Synaptic Vesicles Prepared from Adult, But Not from Embryonic Brain

Neuronal SNARE-proteins such as synaptobrevin, SNAP 25, and synaptotagmin are key players during neurosecretion. So far palmitoylation of SNAP-25 and synaptotagmin 1 have been described in vivo. Here we have analyzed palmitoylation of the SNARE-proteins synaptobrevin 2 and synaptotagmin in vitro using synaptosomal and synaptic vesicle preparations from rat brain. Labeling of synaptic vesicles prepared from adult brain with [3H]palmitate revealed synaptobrevin 2 besides synaptotagmin 1 as major palmitoylated proteins. [3H]Palmitoylation of synaptobrevin 2 was resistant to chloroform/methanol extraction, but sensitive to reducing agents indicating a covalent fatty acid bond to cysteine residues. Palmitoylation of synaptobrevin 2 was also confirmed using endogenous synaptobrevin 2 present in PC-12 cells and synaptobrevin 2 expressed with a vacciniavirus system in Cos cells. In contrast to the situation seen with membrane preparations obtained from adult brain, synaptic vesicles prepared from embryonic rat brain did not support [3H]palmitoylation of synaptobrevin and synaptotagmin. These results suggest, that both synaptobrevin 2 and synaptotagmin were efficiently palmitoylated from mature synaptic vesicles. However, at least one component of the palmitoylation machinery is developmentally upregulated.

The α‐subunits of G‐proteins G12 and G13 are palmitoylated, but not amidically myristoylated

The α‐subunits of the G‐proteins G12 and G13, were expressed with a baculovirus system in insect cells and analysed for acylation. Both proteins incorporated tritiated palmitic and to a lesser extent also tritiated myristic acid. Radiolabel from both fatty acids was sensitive to treatment with neutral hydroxylamine. This result supports a thioester‐type fatty acid bond and argues against amidical N‐myristoylation. Fatty acid analysis after labeling with [3H]palmitic acid showed that palmitate represents the predominant fatty acid linked to Gα12 and Gα13. Separation of cells into cytosolic and membranous fractions revealed that palmitoylated α‐subunits of G12 were exclusively membrane‐bound, whereas [35S]methionine‐labeled proteins were detected in soluble and particulate fractions. Inhibition of protein synthesis with cycloheximide did not block palmitoylation of the α‐subunits. which indicates that palmitoylation occurs independently of protein synthesis.

Timing of palmitoylation of influenza virus hemagglutinin

The timing of the attachment of fatty acids to the hemagglutinin (HA) of influenza A virus was studied. Treatment of virus infected cells with brefeldin A (BFA), a drug which blocks intracellular transport along the exocytic pathway at a pre‐Golgi site, does not prevent palmitoylation of HA. The relationship of HA‐palmitoylation to the oligomerisation and to the proteolytical cleavage of the protein revealed that the uncleaved trimer of HA is the substrate for the acylating enzyme in virus infected cells. The results are discussed with regard to the intracellular site of palmitoylation.