The Art of Protein Sorting: Meeting Report from the Annaberg Meeting, Goldegg, Austria, 9–13 January 2001

The first Annaberg Meeting/EMBO Workshop on ‘Protein Sorting and Processing in the Secretory Pathway’ was held in a small skiing village near Salzburg in 1989. While this conference, attended by about 15 people, was little more than an extended lab meeting of several groups interested in prohormone processing and secretion, the Fifth Annaberg Conference (about 90 attendees) has grown into one of the more significant, enjoyable and intense meetings in protein and vesicle trafficking. As with previous meetings, the attendees were selected not only for their scientific interests but also for their ability to perform on the ski slopes and in the discotheques. A significant theme that emerged from the previous Annaberg Conferences was that inositol phospholipids play a key role in regulating protein and membrane traffic, and several speakers presented evidence demonstrating the importance of these lipids in vesicle trafficking and organelle structure. Pietro DeCamilli (Yale University) showed that a brain-specific form of the enzyme phosphatidyl 4, phosphate 5-kinase, mediates the synthesis of phosphatidyl inositol 4,5 bisphosphate (PtdIns (4,5)P2) on synaptic membranes. PtdIns (4,5)P2 is present at high local concentrations on the plasma membrane, and may be a signal for both the recruitment of clathrin coats and the local polymerization of actin in the endocytic zone to mediate the recycling of synaptic vesicle membrane components. Using latex beads to purify phagosomes, Gareth Griffiths (EMBL) demonstrated that phagosomal actin nucleation in vitro can be regulated by PtdIns (4,5)P2 and sphingosine-1-phosphate as well as by multiple signaling molecules, including PKC and PLD. The structure and function of the Golgi apparatus is also regulated by these lipids: Antonella De Matteis (Consorzio Mario Negri Sud) presented evidence that the beta isoform of phosphatidyl inositol 4, phosphate kinase, which is present on Golgi cisternae and intercisternal spaces, is recruited to the organelle in an ADP ribosylation factor (ARF)-dependent fashion. This enzyme, which synthesizes phosphatidyl inositol 4-phosphate, the immediate precursor to PtdIns (4,5)P2, may be part of a protein complex including Golgi matrix proteins and myosin IIa. Dennis Shields (Albert Einstein College of Medicine) presented evidence that on-going PtdIns (4,5)P2 synthesis is required to maintain the structure of the Golgi apparatus. In its absence the organelle becomes fragmented, in part because a Golgi-specific form of spectrin dissociates from the membrane. Upon resumption of PtdIns (4,5)P2 synthesis, the Golgi apparatus reassembles in a reaction which appears to require clathrin-coated vesicles. Scott Emr (University of California at San Diego), who was one of the first investigators to demonstrate the importance of protein–phospholipid interactions in mediating vesicle trafficking, showed that in yeast cells the formation of multivesicular bodies requires phosphatidylinositol 3-kinase, the product of the VPS34 gene, to generate PtdIns (3)P. Using an elegant genetic selection technique, he demonstrated that the targeting of vacuolar proteolytic enzymes present in the lumen of multivesicular bodies required not only the synthesis of this lipid but also transient ubiquitination. In the absence of ubiquitination, the enzymes remained on the outer limiting membrane of the vacuole. Tom Kirchhausen (Harvard Medical School) described some novel inhibitors that affect different steps in the secretory pathway. One of several interesting compounds imparts a 20 °C block phenotype on cells and could prove to be a powerful reagent in dissecting the formation and function of post-Golgi vesicle trafficking. Scottie Robinson (Cambridge University) described a novel class of coat proteins, GGAs, which are localized to the trans Golgi and trans-Golgi network (TGN). In yeast cells these proteins, which share homology to the ear domain of the γ-adaptin subunit of the adaptor protein (AP)-1 clathrin binding complex, are clathrin associated, and their knock-out affects vacuolar protein sorting and proαfactor processing. In mammalian cells the GGAs and AP-1 complexes may be localized to distinct regions of the TGN, perhaps giving rise to different post-Golgi vesicles. There are numerous examples of high molecular weight coil–coil proteins in the Golgi apparatus and Sean Munro (MRC, Cambridge) demonstrated that some of these possess a ‘GRIP’ motif at their C-terminus which is involved in targeting to the Golgi apparatus. In yeast cells, a GRIP-containing protein, Imh1p, interacts genetically with the GTP-binding protein Ypt6p. Interestingly, knock-out of the gene encoding a GDP–GTP exchange factor for Ypt6p, RIC1, perturbed Imh1p localization to the Golgi apparatus, suggesting that these molecules play a role in the Golgi apparatus, although their function remains to be elucidated. Gary Thomas (Vollum Institute, OR), who first identified the PACS-1 protein, which binds to the phosphorylated cytoplasmic tail of several proteins that reside in or cycle through the TGN, presented evidence that PACS-1 interacts with the AP-1 complex via an 8 amino acid motif. Mutations in this PACS-1 region confer a dominant negative phenotype, which results in disruption of the localization of furin and mannose-6-phosphate receptors. In endocrine cells, the mechanism of secretory granule formation is still unclear. Using an in vitro system that supports homotypic fusion of immature secretory granules, Sharon Tooze (ICRF, London) showed that the t-SNARE syntaxin 6 inhibited vesicle fusion, whereas reagents, including antibodies, to other neuronal v-and t-SNAREs (SNAP 25, syntaxin 1 and VAMP2) had no effect. Additionally, she demonstrated a role for the small GTP-binding protein ARF, which recruits AP-1 to these membranes; the latter also binds the t-SNARE VAMP4. Just how the syntaxin 6 complex and AP-1/VAMP4 may interact in vesicle maturation remains to be determined. Phogrin is a type I membrane glycoprotein, specific to neuroendocrine cell secretory granules and is a member of the tyrosine phosphatase family, although it lacks enzymatic activity. John Hutton (University of Colorado) demonstrated what was a recurring theme: that phosphorylation can regulate protein localization to the Golgi and endosomal compartments. In pancreatic islet β-cells, the trafficking of phogrin to secretory granules is regulated by Ca2+ and cAMP-dependent kinases that phosphorylate specific residues in its cytoplasmic tail. An emerging idea from several talks was that cargo sorting may occur by interaction with specific adaptor molecules localized to different sites in an organelle. Randy Schekman (University of California Berkeley) showed that in vitro generated coat protein (COP)-II vesicles were highly enriched in proαfactor compared to starting microsomes, whereas the nascent vesicle and microsomal phospholipid compositions were virtually identical. In the presence of Brefeldin A (BFA), there was a dramatic decrease in proαfactor incorporation into COP-II vesicles, whereas the same percentage of phospholipid was packaged as in control vesicles. His data are consistent with a receptor model for proαfactor packaging whose sorting into COP-II vesicles is blocked by BFA, whereas phospholipids are packaged via a nonreceptor-mediated process. In a quite different experimental system, Suzanne Pfeffer (Stanford University) presented evidence that the selective sorting of the two different mannose 6-phosphate receptors from endosomes to the TGN (a reaction facilitated by the TIP47 protein that binds to the cytosolic tail of the receptors) may be regulated by Rab 9. In the presence of Rab 9. GTP, the affinity of TIP47 for the receptors was enhanced. Her data suggest that the selective binding of TIP47 to mannose 6-phosphate receptors may be regulated by this small GTP binding protein. Judith Klumperman (University of Utrecht), using cryoimmunoelectron microscopy, demonstrated the existence of two different mannosidase-II-containing COP-I Golgi vesicles: those enriched in either giantin or the KDEL receptor. Little overlap was observed between the KDEL receptor and giantin. These data provide in situ evidence for the occurrence of Golgi-resident proteins in COP-I Golgi vesicles and may indicate the existence of two separate populations of COP-I retrograde vesicles; but whether they have distinct functions remains to be determined. John Bergeron (McGill University) described an interesting cell-free system that supports the formation of a fenestrated tubular network whose morphology resembles that of vesicular–tubular clusters (VTCs). Generation of these structures from purified microsomes required the interaction of p97 and syntaxin 5. His data suggest that tyrosine phosphorylation regulates formation of these structures because phosphorylation of p97 on tyrosine disrupts its binding to syntaxin 5 and inhibits assembly of the network. The concept of a Golgi scaffold or matrix was discussed by Graham Warren (Yale University). He showed that when exit from the endoplasmic reticulum (ER) was prevented, galactosyl transferase recycled to and remained in the ER. In contrast, both GM130 and GRASP65 maintained a perinuclear location and were associated with a large number of small vesicles; Warren suggested that these and other matrix proteins may organize the membrane structure of the Golgi apparatus. In yeast cells, the organization of the Golgi apparatus can be regulated by ARF GDP–GTP exchange factors (GEFs). Catherine Jackson (National Institute of Health) showed that several mutant alleles of the yeast ARF GEF, Gea1 protein, selectively block cargo from the ER or Golgi apparatus, and at the nonpermissive temperature lead to the formation of large tubular networks containing ring-shaped structures. Changes in Golgi morphology are also evident during programmed cell death (apoptosis). Viki Allan (Manchester University) showed that the intermediate chain of the microtubule motor protein cytoplasmic dynein, is cleaved by members of the caspase family of proteases during apoptosis. This results in the loss of dynein from membranes and a decrease in vesicular movement in an in vitro assay. Allan speculated that such a loss of dynein to could contribute to fragmentation of the Golgi apparatus during apoptosis. Peter Novick (Yale University) described his elegant studies on the yeast exocyst, the 8 subunit complex that mediates vesicle tethering to the plasma membrane. The exocyst, together with the v-and t-SNAREs Snc and Sso, respectively, is required for vesicle fusion. Interestingly, the trans membrane domains of these SNAREs are essential for membrane fusion. If these SNAREs are tethered by lipid anchors, exocytosis is blocked at the hemifusion stage, suggesting that the trans membrane domains play a role in the fusion reaction. Given the multitude of SNARE molecules, Richard Scheller (Stanford University) analyzed the specificity of their interactions by comparing conserved and variable motifs that are likely to generate the characteristic four helical bundles in previously cloned as well as in several newly identified SNARE proteins. While multiple Rab proteins provide regulatory elements for SNARE membrane interactions, the mechanism of SNARE pair specificity is still unclear. A novel function for the tetanus-neurotoxin insensitive VAMP was presented by Thierry Galli (Institute Curie). The Ti-Vamp (Vamp7) forms a complex with SNAP-25 and is present in extending neurites and at the plasma membrane, and recycles into a yet-unidentified compartment. In differentiating PC12 cells and hippocampal neurons, Ti-Vamp is required for axon and dendritic outgrowth and differentiation. Regis Kelly (University of California at San Francisco) addressed the question of how plasma membrane components of neuroendocrine cells are recycled selectively via a mechanism termed compensatory endocytosis. He showed that, in part, this may occur by factors that are unique to neuronal cells, in particular a dynamin-binding protein DAP160, a complex polypeptide with multiple binding domains which may stimulate the local organization of the actin cytoskeleton to facilitate endocytosis as well as growth cone growth. Retrograde transport from the plasma membrane to the ER, a pathway used by Shiga toxin, was discussed by Bruno Goud (Institut Curie). Both Rab 11 and 6 regulate this pathway and several Rab 6 isoforms are associated with the Golgi apparatus. Rab 6A (but not the isoform Rab 6A') interacts with Rabkinesin 6, a kinesin-like protein associated with the Golgi apparatus. Rab 5 regulates homotypic fusion of early endosomes; Jean Gruenberg (University of Geneva) presented evidence that an upstream regulator of GTP dissociation inhibitor (GDI) is the protein kinase p38 MAPK, which regulates the activity of Rab 5 by stimulating its removal from endosomes. Changes in p38 MAPK activity in response to different stress-related stimuli alter Rab 5–GDI activity, thereby regulating endocytosis. Hugh Pelham (LMB, Cambridge) presented the Annaberg Lecture, whose theme was disentangling the relationship between the TGN and endosomal compartments and the SNAREs localized to these compartments. He presented genetic and morphological evidence that in yeast cells, transport specificity between the late Golgi and endocytotic compartments is not solely attributable to SNAREs but rather to regulation by Rab proteins and their effectors. In the absence of the reclusive Alfons Grunzelsbacher, who was indisposed, Gareth Griffiths continued a tradition started at the third Annaberg Conference by delivering the Countess Sibilla Feltrinelli Lecture. The main theme of his talk was to understand how the most embarrassing moments in the careers of some cell biologists profoundly affected their scientific creativity (see Figure 1). Competition at the meeting often reached childish levels. Here Graham Warren (left) Hugh Pelham, Randy Schekman and Richard Scheller (right) are about to set out on a mountain hike. Photograph “courtesy” of Gareth Griffiths. The Annaberg Conference is unique. This was particularly evident in the final event of the meeting: an art exhibition bringing together scientists and artists to explore areas of common interest. Many guests from the public also attended this exhibition. Several highly creative and innovative artists, painters, sculptors and photographers presented works related to biology and medicine. Among the art works on display were photographic portraits by Gabi Seethaler, together with the subjects' DNA fingerprints, a series of immunofluorescence micrographs taken by Jennifer Lippincott-Schwartz, and a superb multimedia presentation of the video animation entitled ‘Birth of a Clathrin Coat’ by Allison Bruce and Tom Kirchhausen. This conference was supported by a grant from EMBO and by generous gifts from Novartis Pharma AC and Wyeth-Ayerst Pharmaceuticals.

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