Alpine pathways of membrane traffic
2004; Springer Nature; Volume: 5; Issue: 6 Linguagem: Inglês
10.1038/sj.embor.7400173
ISSN1469-3178
Autores Tópico(s)Bacterial Genetics and Biotechnology
ResumoMeeting Report1 June 2004free access Alpine pathways of membrane traffic Workshop on Protein Sorting in the Secretory Pathway Martin Lowe Corresponding Author Martin Lowe School of Biological Sciences, University of Manchester, The Michael Smith Building, Oxford Road, Manchester, M13 9PT UK Search for more papers by this author Francis A Barr Francis A Barr Department of Cell Biology, Max-Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany Search for more papers by this author Martin Lowe Corresponding Author Martin Lowe School of Biological Sciences, University of Manchester, The Michael Smith Building, Oxford Road, Manchester, M13 9PT UK Search for more papers by this author Francis A Barr Francis A Barr Department of Cell Biology, Max-Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany Search for more papers by this author Author Information Martin Lowe 1 and Francis A Barr2 1School of Biological Sciences, University of Manchester, The Michael Smith Building, Oxford Road, Manchester, M13 9PT UK 2Department of Cell Biology, Max-Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany *Corresponding author. Tel: +44 161 275 5387; Fax: +44 161 275 1505; E-mail: [email protected] EMBO Reports (2004)5:561-564https://doi.org/10.1038/sj.embor.7400173 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info This EMBO workshop was organized by G. Seethaler, D. Shields and S. Tooze, and took place between 13 and 18 January 2004 in Goldegg, Austria, which is a picturesque village close to Salzburg Introduction The Sixth Annaberg Conference continued the success of previous meetings through a combination of interesting talks, lively poster sessions and ample opportunities for the participants to meet informally in the hotel, on the ski slopes and even in the disco. As in previous years, the meeting was held in the medieval Schloss Goldegg near Salzburg, Austria, which provided an unusual but atmospheric setting for a conference dealing with recent advances in cell biology. The presentations covered a wide range of topics relating to protein transport along endocytic and exocytic pathways. In this report, we concentrate on a selection of these talks, and highlight concepts and trends that emerged during the meeting. We apologize to those speakers whose work we were unable to include owing to space limitations. Getting out of the ER… The mechanism by which secretory proteins exit the endoplasmic reticulum (ER) has received much attention in recent years, and our understanding of this process has increased considerably. Many of the key advances have come from the laboratory of R. Schekman (Berkeley, CA, USA), who opened the meeting by describing how the coat-protein complex II (COPII) coat assembles on membranes before vesicle budding. Schekman showed the reconstitution of COPII-vesicle formation using purified components and GTP, rather than the less physiological non-hydrolysable GTP analogues that have previously been used. The trick was to include soluble Sec12, which is the nucleotide-exchange factor for the Sar1 GTPase. This was expected to continually activate Sar1, and, indeed, resulted in stable coat assembly and the production of vesicles that were enriched in cargo proteins. Interestingly, Sec12 was absent from purified vesicles, which led Schekman to propose that it is the ratio of Sec12 to Sec23 (the COPII subunit with Sar1 GTPase-activating protein (GAP) activity) that determines coat stability. He proposed that when the level of Sec12 is high and that of Sec23 is low, as is the case during budding, the coat is stable, whereas in vesicles, the small amount of Sec12 coupled with a large amount of Sec23 promotes uncoating, which allows the vesicles to fuse with the target membrane. A recent morphological study from the group of A. Luini (Santa Maria Imbaro, Italy) has questioned the extent to which COPII vesicles mediate cargo exit at the ER (Mironov et al, 2003). Rather than COPII driving budding, as is commonly thought, Luini and co-workers proposed that it functions only to concentrate cargo for exit in ER-derived tubules. J. Klumperman (Utrecht, The Netherlands) presented recent work aimed at addressing the role of COPII in vivo. By combining electron tomography and a three-dimensional reconstruction with immunogold labelling for COPII, Klumperman showed that COPII is present on vesicular structures that are clearly separate from the ER, which indicates that COPII vesicles are present in cells (Fig 1). Klumperman is now in a position to test whether these vesicles contain cargo by combining labelling for cargo and COPII. Figure 1.Tomogram of a high-pressure frozen HepG2 cell showing a model of a peripheral endoplasmic reticulum exit site. The ER is shown in turquoise, the ribosomes are dark blue and the detached tubulo-vesicular membranes are yellow. The model is projected against a digital section of the original material, in which ribosomes (dark spots) and membrane vesicles are clearly discernible. Figure courtesy of W.J.C. Geerts and A.J. Koster of the Institute of Biomembranes, Utrecht, the Netherlands, and D.G. Zeuschner and J. Klumperman of the University Medical Center, Utrecht, the Netherlands. Download figure Download PowerPoint …and into the Golgi COPI-coated vesicles regulate transport between the ER and the Golgi apparatus, as well as transport within the Golgi apparatus. Several speakers discussed how recruitment of the COPI coat to membranes is regulated. P. Melancon (Edmonton, Canada) focused on Golgi-specific brefeldin-A-resistance factor 1 (GBF1), which is a member of the ADP-ribosylation factor (ARF) guanine nucleotide-exchange factor (GEF) family. In contrast to other ARF GEFs, GBF1 is found on the intermediate compartment and the cis-Golgi. Melancon demonstrated that inhibition of this protein by antibody microinjection resulted in COPI dissociation and the inhibition of transport to the Golgi, which highlights the importance of GBF1 in this transport step. ARFs are also regulated by GAP proteins, and R. Duden (Cambridge, UK) described two new members of this family that are found in mammalian cells: ARFGAP2 and ARFGAP3. Both localize to the intermediate compartment and the cis-Golgi, and are associated with COPI vesicles, probably through direct interaction with the γ-COP-appendage domain, which, as the name suggests, shares structural similarity with the appendage domain of adaptins. Duden proposed the existence of a second binding site on the γ-COP appendage for the α-, β′-, ϵ-COP trimer, which is a coatomer subcomplex that is responsible for the interaction with cargo. Interestingly, Duden showed that the KKXX and KXKXX ER-retrieval signals, which were previously thought to bind COPI by the same mechanism, in fact bind to different subunits: KKXX signals bind to the WD40 domain of the α-COP subunit, whereas KXKXX signals bind to a similar domain in β′-COP (Eugster et al, 2004). Therefore, both the α- and β′-COP subunits of COPI contribute to the recycling of proteins with dilysine signals. The emerging importance of golgins Golgins are a class of coiled-coil proteins that are associated with the cytoplasmic face of the Golgi apparatus. In the Annaberg lecture, G. Warren (New Haven, CT, USA) described several members of this family and their roles in Golgi transport. Previous work in Warren's laboratory showed that the golgins GM130, p115 and giantin can interact to tether COPI-transport vesicles to Golgi cisternae. His recent work has shown that two other golgins, golgin 84 and CASP, interact to form a distinct complex that can also tether COPI vesicles. The reconstruction of vesicle budding and tethering in vitro showed that vesicles tethered by GM130, p115 and giantin contain anterograde cargo but lack Golgi enzymes, whereas vesicles tethered by golgin 84 and CASP are enriched in enzymes but depleted in anterograde cargo. On the basis of these observations, Warren proposed that vesicle-tethering proteins dictate flow patterns in the Golgi stack, with some tethers directing forward transport (for example, GM130, p115 and giantin) and others directing retrograde transport (for example, golgin 84 and CASP). The investigation of other tethering proteins will be necessary to test the generality of this model, but, at present, it is an attractive idea. Interestingly, golgin-84-containing vesicles were found to contain giantin, and vice versa. This might be expected, as tethering proteins must recycle for further rounds of transport. It also implies that vesicle-tethering proteins exist in active and inactive forms that are sorted into separate populations of vesicles. If Warren is correct, understanding these processes will be of great importance in determining how transport through the Golgi is regulated. In addition to vesicle tethering, it is likely that golgins will have other functions and some might have more than one role. This was illustrated by F. Barr (Martinsried, Germany), who showed that GM130 can act as a scaffold for recruitment of the Ste20-like protein kinases YSK1 and MST4 to the Golgi apparatus (Preisinger et al, 2004). These kinases bind to a region of GM130 that is distinct from those involved in vesicle tethering and, at least for YSK1, the interaction induces kinase activity. An important substrate for YSK1 was identified as 14-3-3ζ, which is a signalling protein that is implicated in cell migration. This pointed to a role for Golgi-localized YSK1 in regulating the process, which was confirmed when Barr expressed kinase-defective YSK1 in cells and observed an inhibition of cell migration. He also showed that the Golgi was no longer positioned correctly in these cells, which indicates that YSK1 controls Golgi positioning such that it allows the polarized delivery of membrane to the cell surface. GM130, by recruiting and activating YSK1 in addition to tethering vesicles, might coordinate vesicle transport with the regulatory systems that dictate where newly synthesized proteins should be delivered. The mechanisms underlying the targeting of golgins were discussed by S. Munro (Cambridge, UK). The yeast golgin Imh1 contains a GRIP domain that mediates Golgi targeting through interaction with the small GTPase Arl1, which, in turn, requires Arl3 for its targeting. Targeting of Arl3 is dependent on another protein called Sys1. Co-crystallization of the GRIP domain with Arl1 showed that a conserved tyrosine in the GRIP domain that is essential for targeting fits tightly into a corresponding pocket in Arl1, which provides an explanation for the importance of this residue (Panic et al, 2003). How Arl3 is targeted to the Golgi is less clear. Munro found that this protein is N-acetylated, as are most cytoplasmic proteins in eukaryotes, and identified N-terminal acetyl-transferase complex C (NatC) as the enzyme responsible (Behnia et al, 2004). Abrogation of NatC activity not only prevented Arl3 acetylation, but also blocked its targeting to the Golgi, which resulted in a loss of Imh1 recruitment. These findings indicate a new function for N-acetylation and raise the possibility that other proteins also use this modification for membrane targeting. Membrane fusion It has been known for several years that protein palmitoylation has an important role in membrane-fusion reactions, although the molecular details have remained elusive. By studying the fusion of yeast vacuoles, the group of C. Ungermann (Heidelberg, Germany) discovered a mechanism for palmitoylation and identified how it might regulate membrane fusion. They were able to show that, surprisingly, the SNARE Ykt6 can catalyse palmitoylation of the fusion factor Vac8, and that this activity is essential for vacuolar fusion (Dietrich et al, 2004). Palmitoylation required the ATPase Sec18, which indicated a new priming role for this protein in addition to its ability to break up SNARE complexes. Ykt6 is a highly conserved SNARE that is required for fusion at the Golgi apparatus, endosomes and vacuoles. This led Ungermann to speculate that it might have a general role in catalysing the acylation of fusion factors during membrane fusion. Related SNAREs might be able to acylate proteins at other compartments. Ungermann pointed out that the N-termini of Ykt6 and Sec22, which is an ER-to-Golgi SNARE, share the same structure. This is the region in Ykt6 that is responsible for the palmitoylation of Vac8, which implicates Sec22 in a similar reaction during ER-to-Golgi transport. Lipids and the regulation of endosome function The regulation of organelle motility is one of the least understood aspects of membrane traffic, and some exciting progress on this front was reported at the meeting. M. Zerial (Dresden, Germany) talked about the role of Rab5 as a regulator of early endosome function, and showed how this is tightly coupled with phosphoinositide metabolism. The addition of Rab5 to endosomes stimulates both membrane fusion and the generation of phosphatidylinositol-3-phosphate (PtdIns3P), which is important for the recruitment of a range of effector proteins that are needed for endosome tethering and motility. One aspect of this motility is negative regulation through anchoring to the actin cytoskeleton, which is a process involving Rho GTPases and formins. Another aspect is the positive control of microtubule-based motility by Rab5 and PtdIns3P, which points to the involvement of a kinesin motor protein that could be regulated by binding to phosphoinositides. A bioinformatics approach resulted in the identification of a candidate motor that fulfilled these criteria. The link to the control of endosome motility was confirmed using inhibitory antibodies to the motor domain and the expression of dominant-negative mutants. It has also been reported that Rab27A and Rab6 are regulators of vesicle motility. Together, these findings establish the theme that Rab GTPases are regulators of both membrane–membrane and membrane–cytoskeleton tethering events. Another aspect of lipid function in the control of endosome function was presented by J. Gruenberg (Geneva, Switzerland), who talked about the role of lysobisphosphatidic acid (LBPA) and the protein ALIX in the formation of multivesicular bodies (MVBs) in the late-endocytic pathway (Fig 2; Matsuo et al, 2004). His group attempted to reconstitute some aspects of the MVB-formation pathway using liposomes made with LBPA and an acidic luminal pH. These liposomes spontaneously form internal vesicles that are similar in appearance to MVBs, which indicates that LBPA self-associates at low pH and drives this process. To search for regulators of this event, Gruenberg and co-workers examined which proteins could bind to such liposomes and identified ALIX, which is a homologue of the class E vacuolar protein-sorting mutant Bro1. ALIX regulates the formation of MVB-like liposomes in vitro, and the organization of LBPA-containing endosomes in vivo. Figure 2.Fluorescence-microscopy image of liposomes containing lysobis-phosphatidic acid. Liposomes with an internal acidic pH were prepared with LBPA and incubated with the fluorescent lipid-binding dye FM2-10 before analysis using fluorescence microscopy. Note the presence of numerous internal vesicles, which are similar to those found in multivesicular bodies in cells. (Copyright: J. Gruenberg of the Department of Biochemistry, University of Geneva, Switzerland.) Download figure Download PowerPoint Membrane-protein sorting between Golgi and endosomes The sorting of membrane proteins was one of the main themes of the meeting, and was touched on by several speakers describing work in both yeast and mammalian cells. One important aspect is the quality control of membrane-protein complexes, which is a process that the group of H. Pelham (Cambridge, UK) has previously shown to be mediated through the recognition of aberrant transmembrane domains (TMDs) by a ubiquitin ligase Tul1—itself a multi-spanning membrane protein. Tul1 interacts with the TMDs of its targets and ubiquitylates them, which results in targeting to the vacuole. However, this is not the whole story, as Tul1 is found only in plants and fungi, and there are proteins that do not rely on Tul1 for vacuolar targeting. The search for a second pathway resulted in the identification of a bi-functional complex of the transmembrane protein Bsd2 and the HECT-domain ubiquitin ligase Rsp5 (Hettema et al, 2004). Together, these two proteins function in the same manner as Tul1, although with different substrate specificity. The group of S. Emr (La Jolla, CA, USA) also identified Rsp5 in a genetic screen, and showed that the sorting of proteins from the delimiting membrane of the vacuole to intraluminal vesicles is compromised in Rsp5 mutants (Katzmann et al, 2004). Bsd2 and Rsp5 are also conserved in higher eukaryotes that lack Tul1 and, as Emr pointed out, related HECT-domain ubiquitin ligases are known to function in receptor downregulation in the transforming growth factor-β and Notch signalling pathways, which neatly rounded off this story. Coat proteins have an important function in protein sorting between the Golgi apparatus and lysosomes, which was addressed by J. Bonifacino (Bethesda, MD, USA). A bewildering array of coat proteins and associated adaptors might function at this stage, including clathrin and its AP1 adaptor, the GGA proteins and the retromer complex. The Bonifacino group has now tried to pinpoint the exact steps at which these proteins are required. They first looked at the GGA proteins that bind to the small GTPase ARF1 and clathrin, and are present on the Golgi apparatus. Knockdown of the GGA proteins causes the secretion of lysosomal enzymes, probably due to the missorting of the cation-independent mannose-6-phosphate receptor (CI-MPR) in the trans-Golgi network (TGN). M. Lowe (Manchester, UK) presented evidence that this process might be regulated by the lipid phosphatase OCRL1, which binds to the clathrin heavy chain and localizes to the TGN and CI-MPR-positive vesicles. Expression of a dominant-negative phosphatase-dead form of OCRL1 results in a loss of GGA proteins from the TGN and the missorting of the CI-MPR. By contrast, the retromer is present on early endosomes, similar to the transferrin receptor, and requires PtdIns3P to localize to these membranes. Depletion of the retromer subunit Vps26 causes a decrease in the levels of the cation-independent MPR owing to a failure to recycle it back to the TGN, which results in its degradation in the lysosomes (Arighi et al, 2004). This is similar to the phenotypes of yeast-retromer mutants reported by the Emr laboratory. Linking membrane transport and cell death Phosphofurin-acidic cluster sorting protein 1 (PACS1) is another protein that is involved in endosome-to-TGN transport. It binds to cargo proteins that contain acidic clusters and sorts them into endosome-derived transport carriers. G. Thomas (Portland, OR, USA) introduced a related protein, PACS2, which functions as a cargo sorter in the early secretory pathway. PACS2 can bind to both COPI and a subset of ER proteins that have acidic clusters in their cytoplasmic tails, mediating their inclusion in retrograde transport vesicles. The expression of mutant PACS2 or depleting the protein resulted in the escape of these proteins to the cell surface. Unexpectedly, the depletion of PACS2 also fragmented mitochondria. This was attributed to the mislocalization and cleavage of the pro-apoptotic ER protein BAP31, which is known to fragment mitochondria and trigger apoptosis. Surprisingly, however, PACS2-depleted cells failed to enter apoptosis. Further studies showed that PACS2 itself is an important pro-apoptotic regulator, which targets the Bcl2 family member Bid to mitochondria. PACS2 therefore seems to have a dual role, acting both as a cargo sorter in the early secretory pathway and as a pro-apoptotic regulator. As such, it is ideally placed to act as a link between sensing stress in the early secretory pathway and translating this into initiation of the apoptotic programme. Future perspectives In recent years, much of the molecular machinery that underpins membrane transport along the endocytic and exocytic pathways has been identified. At this meeting, it became evident that we are making rapid progress in understanding the mechanisms by which these proteins function. It was also apparent that we are beginning to appreciate the diverse roles of lipids in regulating transport. We anticipate that rapid progress will continue in these areas, driven by further technological and conceptual advances. In all respects, this was an excellent meeting, and much credit should be given to the organizers who achieved a successful blend of talks, posters and informal gatherings. Let us hope that there will be more EMBO-funded Annaberg Conferences in the future. Acknowledgements We thank all of the speakers mentioned for their corrections to the manuscript, and S. Munro for suggesting the title. Biographies Martin Lowe Francis A Barr References Arighi CN, Hartnell LM, Aguilar RC, Haft CR, Bonifacino JS (2004) Role of the mammalian retromer in sorting of the cation-independent mannose 6-phosphate receptor. 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