We have recently succeeded in reconstituting fusion with all-purified components!! In these studies, fusion requires the Ypt7p GTPase, the HOPS complex, all 4 SNAREs, SNARE chaperones Sec18p and Sec17p, ATP, and the regulatory lipids (ergosterol, diacylglycerol, and phosphoinositides). This reconstitution is a remarkable advance, and provides an opportunity for us to study how these factors work together.
Several aspects of our system are noteworthy:
- Our assays are colorimetric and flourimetric, and hence quantitative and rapid. It can be done with pure frozen components.
- The indicated components are largely purified and in the freezer,
along with antibodies or other inhibitors.
- The biochemically defined early subreactions provided a unique
opportunity to solubilize and reconstitute the every stage of the
- Genomics and proteomics have now moved to the forefront! We have
screened the collection of 6500 yeast deletions for fragmented vacuoles
(the phenotype of blocked fusion) and it's yielding a cornucopia of new
proteins involved in the reaction, from kinases to the vacuolar H+ -
ATPase to many proteins of unknown function. We are making recombinant
forms of each, raising antisera, and exploring how each contributes to
the overall reaction.
- Exploiting molecular tagging with green fluorescent protein and
high-tech microscopy, we have discovered a novel "ring" of fusion
factors surrounding the touching membranes of each pair of apposed
vacuoles, and can study how this ring assembles and leads to fusion.
- Each facet of the fusion reaction can be studied in vivo, exploiting
the ease of vacuole visualization and the power of yeast genetics, in vitro
through exploitation of our many assays for stages of the fusion of purified
vacuoles, and with reconstituted liposomes and all-purified components.
For an overview of this system, see Wickner and Haas (2000) Ann Rev.
Biochem. 69, 247-275, Wickner (2001) EMBO J., 21,
1241-1247, and Wickner (2010) Ann Rev. Cell Dev Biol 26, 115-136.
It's Not Just a Job
OK, what may also be evident is that I'm quite in love with this work!
Science is fun! Always working with young people from all over the world is
stimulating and enjoyable! Well, I have always felt very very lucky to have a
lab of my own, and have done my very best to help my postdocs and students
achieve that goal too. As much as I enjoy and derive satisfaction from the
science, I've been gratified to see how the many alumni of the lab have achieved
spectacular success on their own (see the lab
A Little About Us
The lab is small but excellent. It consists of several postdoctoral fellows, 2 research associates, and me. Our lab itself is spacious and lovely in an old
brick building in a beautiful, beautiful
part of the world. Hanover, NH is a New England collegetown, home to a
small top-quality, Ivy-League university (Dartmouth). It has none of the
crime and problems that plague the big cities of the US and their mega-universities,
yet is just two hours from Boston, 4.5 from NY, 3 from Montreal, and 5
from Quebec. This town is quite atypical, for it has several restaurants
with "continental cuisine", two Chinese restaurants, an Indian one, a
Japanese sushi bar, an excellent bookstore, a bagel shop, ... . One of
the major attractions is the outdoor life--this is the perfect spot for
hiking, canoeing, skiing, etc., and most folks here work hard in the lab
and then go enjoy the outdoors.
To really get into what the lab is doing, here are two further resources,
"Projects" and "Membrane Fusion". The first will provide an in-depth review
of the tools at our disposal, what the lab folks are doing now, and projects
we've dreamed of doing in the future. The second provides an overview
of the membrane fusion literature, updated every year or two.
Here's an overview of the lab's science, in 3 sections. The first is
a summary of who we are, and what tools we have at our disposal. The second
is a listing of what each lab member is working on. The third is a random
list of projects we've yet to tackle.
Lab folks: Bill, Amy, Deb, Hong-ki, Michael
Genetics, incl. deletion screen; t.s. screen?
Lipid analysis; MS of deacylated? PLD to IPx to MS?
Ca flux measurements
Reconstituted assays & docking and fusion, with pure compenents
ATP: Sec18, Vps33, Vps11, V1V0, PI4K, PI4P5K, G to F actin, GTP
pool, protein kinases, lipid kinases, yet the reaction goes fine with
apyrase after priming!
GTP: Analysis of bound nucelotide by pull-down, PCA extraction, MS?
Soluble factors: Sec18p, Sec17p, kinases, HOPS, PI(4,5)P2, Vam7
Reversible: Minus ATP, excess Sec17p, anti-Sec18p, anti-Ypt7p,BAPTA, GTPgS,
neomycin (with frozen vac's),
phospholipase C, anti-PIP(2), PX, anti-Vam7p, dilution, C1B domain, latB,
Gyp7 (by Vam7)
Irreversible: anti-Vam3p, anti-other SNAREs
Unclear if reversible: anti-HOPS subunits, excess cytosol, high salt,
low salt, jasplakinolide, wortmanin, phosphoinositide ligand domains,
Fusion (2 assays!)
cis-SNARE complex; co-IP
cis-SNARE complex disassembly on the vacuole, on "beads"
LMA1 binding to vacuoles, to pure Sec18p
Docking (visual assay)
Vertex enrichment of factors
Resistance to each inhibitor; kinetics, or staging
Calcium release upon docking
65S to 38S
HOPS binding to Ypt7p
Release from the vacuole of:
GTP hydrolysis by GST-Ypt7p
Pure protein reagents:
PX, FYVE, and other lipid-binding domains
Vam3 soluble domain
IB2, Trx, Sec17, Sec18, 5 SNAREs (protein, peptide, domain), Ypt7p, Gdi1p,
Cdc42p, Rho1p, Las17p, Rom2p, Cla4p, Plc1p, Vps 11, 16, 18, 33, 39, 41,
Pep4, Pho8, Vac8, Act1, Vrp1, Arp3, Glc7, Cmd1, Vtc1, CPY, Ykt6
Lab Directions, January 2016
A. How does Sec17p trigger fusion?
1. What lipid headgroups and fatty acyl chains determine this requirement?
2. Is the need for Sec17p greater or less at limiting Ypt7, SNAREs, and/or HOPS?
3. Does Sec17 addition affect the fusion vs lysis balance?
4. Does added Sec17p displace HOPS from the SNAREs (see also Amy's floatation data)
5. Synthetic phenotype in vivo of the Sec17-FSMS mutant with various Ypt7, SNARE, and HOPS subunit mutants
6. Will Sec17 addition overcome the block to fusion caused by the absence of DAG, Erg, and PE?
7. In the absence of HOPS, where 3Q + 1R gives moderate trans-SNARE levels but very little fusion, will Sec17 addition trigger fusion?
B. HOPS catalysis of trans-SNARE complex assembly
1. Do low concentrations of Sec17p stimulate further?
2. Why/how is fusion of 2Q+1R+ 1nM Vam7 only stimulated by Ypt7:GTP on the 1R?
3. Amy's assay: floatation of 1R-RPLs that were incubated with 3sQ's + HOPS shows dependence on each for floatation of all.
a. What determines the background; swing-out vs fixed angle spins
b. Do only 1R-RPLs bind HOPS, or why else is the 1R "special"?
c. Is the product 4SNARE complex, 4SNARE complex bound to HOPS, or HOPS with each of the 4 uncomplexed SNAREs bound?
d. At high levels of 1R and of the soluble 3Q's, and with longer times, is there more SNARE complex that floats than HOPS that floats?
4. At 1:32,000 SNAREs (1R/3Q), HOPS supports fusion much better than 2% PEG, even though both work fine at high SNARE levels. Since there's no Sec17/18, and no free Vam7 to be recruited, can one use this to demonstrate a 4th function of HOPS, namely to catalyze trans-SNARE complex assembly?
C. Fusion vs Lysis
1. Lysis only follows fusion
2. Is lysis:fusion altered at low SNAREs, Ypt7, with P-HOPS, or with Sec17?
D. Better reagents: Improved purifications of all but Vam7 for purity, mono-disperse, without tags
E. Assays to be developed:
1. FRET of trans-SNARE pairs
a. Derivatize Vam3 and Nyv1 on random Lys with FRET pairs
b. Unique cys constructs
2. C-Laurdan or other probes of non-bilayer structure
3. Is leakiness, as per Zucchi and Zick, a measure of forming nonbilayers which might be assayable by MuNaNa entry to entrapped enzyme and does this respond to various parameters of fusion intermediates? Is it seen with trans-SNARE paired 3Q:1R without HOPS, or without DAG,Erg,PE, or under conditions without fusion that can be triggered by Sec17?
The Great Search for Other Protein Factors:
a) published proteins: LMA1, actin, Vam10, Rho1, Cdc42p, Calmodulin, PP1A, Vo, Vrp1p, Vps1p, Ccz1/Mon1p, Vtc1-4p, Eno1p, Plc1p, Bem1p
b) Cloning, expression, Ab, and inhibition tests for:
a) Ccz1; see Klionsky work, and J Cell Sci 114, 3137 (2001)
b) Cdc10; a "hit" in our screen, and see Nature Neurosci 2, 434 (1999)
c) Other intriguing hits from the screen, all strongly B/C:
NAT3, protein N-acetyl transferase
YAL026, P-type Ca ATPase (our reaction is sens to thapsigargin)
TOR2; some inhibition by N-terminal Ab
BMH1,2; 14-3-3 proteins, involved with TOR pathway, perhaps with PKC regulation. Each gives strong B/C phenotype upon deletion.
LUV1 (VPS54)/RIC1; a complex that's an exchange factor for Ypt6 (which, like these, gives a good class B phenotype upon deletion).
YDR200C; 60%B, 0-20% fusion in vitro (Masashi). No expression of pMBP or MBP fusions
YNL080C; only 2 in vivo fusions in 3' (Masashi; this is very low). 100%C to 10%B. 15-40% of w.t. fusion in vitro. No expression obtained by MBP or pMBP fusions.
YLR091; 90%C, in vitro fusion not determined
SAC7; GAP for Rho1
ACFII; cortical actin assembly
SACII/VPS52; part of VPS52, 53, 54 complex, 100%B/C
GIM1, tubulin and actin chaperone
BIK1; a MAP; 100%II
BUB3; microtubule-loss checkpoint
CIN1; microtubule stability
EHT1, alchohol acyltransferase (100%C!)
IPK2; inositol polyphosphate kinase (100%B)
ISC1 as well as PLC1
LCB5 (100% B), long chain base kinase
INP54; inositol polyphosphate 5'-phosphatase
GTPases and effectors:
ROM2 (GEF for Rho1; Ab inhibits the reaction!!)
BEM1 (binds Cdc24, the GEF for Cdc42p)
BEM2 (GAP for Rho1, but weak phenotype)
BEM4 (100%B); effector of Rho GTPases
VPS1; modest phenotypte, vps1D vacuoles don't fuse in vitro, but our Ab doesn't inhibit; Andreas says his Ab does inhibit. Kevin worked on this here...
ARF1; GTPase, strong phenotype
TPD3/FUN32; PP21-regulatory subunit of ceramide-activated
STE4, subunit of a trimeric GTPase
GLO3; GAP for Arf1,2
LTE1; 50% C, GNEF for an unknown target
YBR025C; 100% B/C!. An unknown GTPase
ELM1; 100% B/C, ser/thr protein kinase
CLA4; actin-regulatory. Does Ab inhibit?
MCK1; ser/thr kinase; 100% B/C
CKA2; catalytic subunit of caseine kinase 2; 70%B/C
YBR028c; weak phenotype
ALK1; 100%B/C, ser/thr kinase
CTK1/SNB32; 90% B/C
GIN4/ERC47; ser/thr kinase; 60% B/C
SNFI; 100%B/C; ser/thr kinase
STE11; 100% B/C; MAPKKK
RIM 15; 60%B/C; ser/thr kinase
DIG1; 100%B/C; MAP kinase assoc protein
YAL040c; G1/S specific cyclin; 100%B/C
CLG1; 100%B/C; cyclin with Pho85
PTC1; PP2C family (30%B)
MSG5; protein tyrosine phosphatase (50%B)
Regulatory factors for PP1:
SHP1 (100% B/C)
REG2 (70 % B)
Antibodies that inhibit that need pursuit:
a) Vps45. Gary (08/23/00) and Masashi (1/22/03) saw that anti-Vps45, IgG or affinity-purified, inhibits while the control IgG and heat-killed do not. Might it be Ab to an epitope shared with Vps33? Or, the real McCoy? On vacuoles by Masashi's westerns.
b) Vps5; Masashi finds that the Ab inhibits, and the effect of vps5D on fusion, in vivo and in vitro, is not just due to lack of Vam10p. On vacuoles by Masashi's blots.
c) YLR119, YLR201, YDR065; Ab's from Gary. Both Gary and Masashi saw inhibition.
d) Sys6/Imh1; found with HOPS, deletion causes fragmentation, antibody inhibits
Alumni remaining notes:
Antibodies now being purified and tested; * means no Ab inhibition:
YOR068C; 90%B. Manuscript well-along. Needs fusion assays where only Vam10p is missing.
YBR174C--70%B; 5% fusion in vitro; excellent expression of a pMBP fusion; not in table 9 (phenotype went away with time!)*
YDR136C--60%B to 100%C; no fusion in vitro; no expression of full-length pMBP or MBP fusions, but truncated version made and sent off to rabbitland (not in table 9)*
YPL055C/LPE17 and YBR174C; 50% B; del pep4, pho8 vac's don't fuse; poor expression of pMBP or MBP fusions, but excellent expression of a his-tagged construct*
YOR359w--6/3', 60%B. No expression of pMBP fusions, but truncated version made & sent off.*
YDR433w--15/3', 50%B; no fusion in vitro. Good expression of an MBP fusion.* No antibody inhibition. However, even 80ng/ml of 433 gives good vacuole fusion stimulation.
YER083c--8/3'; 50%B. Doesn't fuse in vitro. No expression of MBP or pMBP fusions, but truncated version worked & sent off.*
[YGL223C--9/3', 90%B; fuses fine in vitro]*
YLR025W/VPS32--100%C to 60%C; no fusion in vitro. Not in table 9. *
YLR091W--4/3', 90%C. Good expression of a pMBP fusion.*
YLR260W--50-100%C; 20% of wt fusion in vitro.*
YMR032W--80%BC to 100%C. No expression of MBP or pMBP fusions. Not in table 9.
Osmostress-induced fusion is fine in vivo.*
YDR200C--60%B; 0-20% fusion in vitro. No expression of pMBP or MBP fusions.
[YLR261C--6/3', 60-90%B; fuses fine in vitro]
YML013C-A--20%B to 40%C. Good expression of a pMBP or MBP fusion!*
[YMR269W--100%B to wt!; fuses fine in vitro]
YNL080C--2/3', 100%C to 10%B!; 15-40% of wt fusion in vitro. No expression of MBP or pMBP fusions.
In table 9, but not on the above list; had phenotypes other than largely B, C:
*No inhibition of fusion by antibodies...
How about two others:
YML013; 35% B,C, 30% in vitro fusion rate, 6/20 osmotic fusion response
YLR091; 90%C, in vitro fusion not determined.
1. YDR200C; 60%C, almost no in vitro fusion. Also, YNL080C; scored as 100%C or as 10%B, reduced in vitro fusion. Neither was expressable as a pMBP fusion; try yeasts, insect cells?
2. YLR091; 90%C, 6/20 osomotic stress fusion, in vitro fusion N/D. Make fusion protein & Ab!!
Project Ideas for Vidya (Aug 10, 2009)
1. Content mixing assay.
2. Assay of hemifusion, using lumenal NBD dequenching (after dithionite treatment) plus R18 (or some such added fluorophore). Kevin showed that R18 dequenching and resistance to Ab to Vam3p occurred together and rapidly in vacuole fusion reactions synchronized by withholding Vam7p, whereas content mixing was slow. Can this be seen with RPLs?
3. HOPS higher overexpression, using either Picchia expression or multi-copy Gal in S. cerevisiae.
4. Linker-scanning mutagenesis of each HOPS subunit, scoring for a) HOPS subunit composition, b) fusion, c) PIPx binding, d) Vam7p binding, e) Ypt7p:GTP binding, f) Ypt7p nucleotide exchange. Study of which activities are needed for tethering, vertex ring enrichment, trans-SNARE assembly, fusion.
5. "Do a Pollard!" Kd, K+1,... for subreactions and subsets of the components. Does Ypt7p binding to HOPS alter its other affinities? Measure the mutual binary associations of Vam7p, PI3P, and HOPS, then ask whether the presence of all 3 gives a far higher affinity. Does Sec17p interfere?
6. FRET assays with Cy3-FYVE and Cy5-FYVE for PI(3)P enrichment in a microdomain.
7. Kai's lipid phase monitor fluorophores.
8. PIPx is needed for SNARE chaperone synergy; is this true with Vam7p-SNARE domain only (no PX domain)?
9. Direct octylglucoside-SNARE addition to reverse-phase LUVs.
An Outline of the Membrane Fusion Literature
Prof. Charlie Barlowe (down the hall) and I teach a seminar course for
1st and 2nd year grad students every other year. Here is the outline of
my sessions, which presents the membrane fusion literature, updated as
of the winter of '03.
I. Assays of membrane fusion and methods for its study
Biochemical Approaches; for a full review of cell-free fusion, see Jahn et al. (2003) Cell 112, 519. A succint review, focused on assays per se: Cook and Davidson (2001) In vitro assays of vesicular transport. Traffic 2, 19.
A. Golgi Compartment mixing assay
1. Fries and Rothman (1980) Transport of vesicular stomatitis virus glycoprotein in a cell-free extract. PNAS 77, 3870.
2. *Balch et al. (1984) Reconstitution of the transport of protein between successive compartments of the Golgi measured by the coupled incorporation of N-acetylglucosamine. Cell 39, 405.
B. ER to Golgi assay
1. Baker et al. (1988) Reconstitution of SEC gene product-dependent intercompartmental protein transport. Cell 54, 335.
2. Lapushin et al. (1996) Biochemical requirements for the targeting and fusion of ER-derived transport vesicles with purified yeast Golgi membranes. J. Cell Biol. 132, 277.
3. Barlowe (1997) Coupled ER to Golgi transport reconstituted with purified cytosolic proteins. JCB 139, 1097.
C. Consilience; A mammal is just a hypo-evolved yeast! Dunphy et al. (1986) Yeast and mammals utilize similar cytosolic components to drive protein transport through the Golgi complex. PNAS 83, 1622. Also, 10 Wilson et al. (1989) A fusion protein required for vesicle-mediated transport in both mammalian cells and yeast. Nature 339, 335.
D. Vacuole fusion; in color
1. Review; Wickner (2002) Yeast vacuoles and membrane fusion pathways. EMBO J. 21, 1241.
2. Conradt et al. (1992) In vitro reactions of vacuole inheritance. JCB 119, 1469; Haas et al. (1994) G-protein ligands inhibit in vitro reactions of vacuole inheritance. JCB 126, 87. Most recently, see Wang, L., Seeley, S., Wickner, W. and Merz, A. (2002) Vacuole fusion at a ring of vertex docking sites leaves membrane fragments within the organelle. Cell108, 357; Seeley, E.S., Kato, M., Margolis, N., Wickner, W., and Eitzen, G. (2002) The genomics of homotypic vacuole fusion. Mol. Biol. Cell 13, 782-794; Eitzen, G., Wang, L., Thorngren, N., and Wickner, W. (2002) Remodeling of organelle-bound actin is required for yeast vacuole fusion. J. Cell Biol. 158, 669; Wang, L., Merz, A., and Wickner, W. (2003) Hierarchy of protein assembly at the vertex ring domain for yeast vacuole docking and fusion. J. Cell Biol. 160, 365; Wickner, A.J. and Merz, A (2004) Resolution of organelle docking and fusion in a cell free assay. PNAS 101, 11548; Fratti et al. (2004) Interdependent assembly of specific regulatory lipids and membrane fusion proteins into the vertex ring domain of docked vacuoles. J. Cell Biol. 169, 1087.
E. Purification of proteins by functional assays:
1. NSF; Block et al. (1988) Purification of an N-ethylmaleimide-sensitive protein catalyzing vesicular transport. PNAS 85, 7852.
2. SNAP; Clary and Rothman (1990) Purification of three related peripheral membrane proteins needed for vesicular transport. JBC 265, 10109.
F. Assays of physical association
1. Zerial and the Rab effectors; Christoforidis et al. (1999) The Rab5 effector EEA1 is a core component of endosome docking. Nature 397, 621.
2. Sollner et al. (1993) SNAP receptors implicated in vesicle targeting and fusion. Nature 362, 318.
3. Seals et al. (2000) A Ypt/Rab effector complex containing the Sec1 homolog Vps33p is required for homotypic vacuole fusion. PNAS 97, 9402.
4. Guo et al. (1999) The exocyst is an effector for Sec4p, targeting secretory vesicles to sites of exocytosis. EMBO J. 18, 1071.
5. Sacher et al. (1998) TRAPP, a highly conserved novel complex on the cis-Golgi that mediates vesicle docking and fusion. EMBO J. 17, 2494.
7. Burd et al. (1997) A novel Sec18p/NSF-dependent complex required for Golgi-to-endosome transport in yeast. Mol. Biol. Cell 8, 1089.
8. Synaptic components and associations. Succinct review: Mochida (2000) Protein-protein interactions in neurotransmitter release. Nueroscience Research 36, 175. Also, for basic relations between docking, priming, and fusion, in PC12 cells (a neurobiologists vacuole) and other systems, see Klenchin and Martin (2000) Priming in exocytosis: Attaining fusion-competence after vesicle docking. Biochimie 82, 399-407. More comprehensive reviews: 20 Calakos and Scheller (1996) Synaptic vesicle biogenesis, docking, and fusion. Physiol. Rev. 76, 1. Also, Sudhof (1995) The synaptic vesicle cycle: a cascade of protein-protein interactions. Nature 375, 645. Data indicating that docked synaptic vesicles may not be trans-SNARE paired is in Hayashi et al. (1994) Synaptic vesicle membrane fusion complex: action of clostridial neurotoxins on assembly. EMBO J. 13, 5051 and Broadie et al. (1995) Syntaxin and synaptobrevin function downstream of vesicle docking in Drosophila. Neuron 15, 663 question the role of SNARE pairing in fusion even more profoundly. The readily-releasable pool of vesicles may not even be docked or tethered!--Rizzoli and Betz (2004) The structural organization of the readily releasable pool of synaptic vesicles. Science 303, 2037.
A. sec mutant selection of Schekman
1. *Novick and Schekman (1979) Secretion and cell-surface growth are blocked in a termperature-sensitive mutant of Saccharomyces cerevisiae. PNAS 76, 1858.
2. Novick et al. (1980) The identification of 23 complementation groupsrequired for post-translational events in the yeast secretory pathway. Cell 21, 205.
3. Kaiser and Schekman (1990) Distinct sets of SEC genes govern transport vesicle formation and fusion early in the secretory pathway. Cell 61, 723. A lovely use of epistasis and synthetic lethality to order a pathway.
B. vps mutant selection.
1. Rothman and Stevens (1986) Protein sorting in yeast: mutants defective in vacuole biogenesis mislocalize vacuolar proteins into the late secretory pathway. Cell 47, 1041.
2. Banta et al. (1988) Organelle assembly in yeast: characterization of yeast mutants defective in vacuolar biogenesis and protein sorting. JCB 107, 1369.
3. 30 Kitamoto et al. (1988) Mutants of Saccharomyces cerevisiae with defective vacuole function. JBact 170, 2687.
And, also, there were the pep mutants of B. Jones, the end mutants of Riezman, and the vac mutants of Weisman, many of which were all in the same set of genes as ("allelic" with) the above 3 sets.
4. Morphology can help organize the genetics; see ref. D2 above, and Raymond et al. (1992) Morphological classification of the yeast vacuolar protein sorting mutants: Evidence for a prevacuolar compartment in class E vps mutants. MBC 3, 1389.
C. 2-hybrid; Stenmark et al. (1995) Rabaptin-5 is a direct effector of the small GTPase Rab5 in endocytic membrane fusion. Cell 83, 423.
Crystallography (see II3 below) and fluorescence light microscopy (Wang et al., 2002; ibid, 2003; Roberts et al. (1999) Endosome fusion in living cells overexpressing GFP-rab5. J. Cell Sci. 112,3667).
Liposome fusion as a model of biomembrane fusion (also see Section IV, for phosphoinositides, which may form microdomains together with ergosterol and DAG, as in Fratti et al. (2004) Interdependent assembly of specific regulatory lipids and membrane fusion proteins into the vertex ring domain of docked vacuoles. JCell Biol. 167, 1987). Review--Chernomordik & Kozlov (2003) Protein-lipid interplay in fusion and fission of biological membranes. Ann Rev Biochem 72, 175-207.
A. The basic assays are fluorescent; Wilschut et al. (1980) Kinetics of calcium ion induced fusion of phosphatidylserine vesicles followed by a new assay for mixing of aqueous vesicle contents. Biochem. 19, 6011.
B. The effects of osmotic and physical stress. Malinin et al. (2002) Osmotic and curvature stress affect PEG-induced fusion of lipid vesicles but not mixing of their lipids. Biophys. J. 82, 2090. Outer and inner leaflets don't mix synchronously--Lee and Lentz (1997) Evolution of lipidic structures during model membrane fusion and the relation of this process to cell membrane fusion. Biochem. 36, 6252-9. Fusion and lysis don't always respond the same to lipid changes--Haque et al. (2001) Influence of lipid composition on physical properties and PEG-mediated fusion of curved and uncurved model membrane vesicles: "Nature's Own" fusogenic lipid bilayer. Biochem. 40, 4340-8. Fusion is always accompanied by lysis--Burgess et al. (Lentz) (1992) Modulation of poly(ethyleneglycol)-induced fusion by membrane hydration; importance of interbilayer separation. Biochem. 31, 2653-2661. Effects of tension--Markosyan, Melikyan, and F.S. Cohen (1999) Tension of membranes expressing the hemagglutinin of Influenza virus inhibits fusion. Biophys. J. 77, 943-52.
C. Divalent cation-induced fusion; Ca>Mg in general, and it's all very dependent on the specific lipids. Bentz et al. (1983) Kinetics of divalent cation induced fusion of phosphatidylserine vesicles: correlation between fusogenic capacities and binding affinities. Biochem. 22, 3320-3330; Bentz and Duzgunes (1985) Fusogenic capacities of divalent cations and effect of liposome size. Biochem. 24, 5436; Leventis et al. (1986) Divalent cation induced fusion and lipid lateral segregation in phosphatidylcholine-phosphatidic acid vesicles. Biochem. 25, 6978; Stamatatos et al. (1988) Interactions of cationic lipid vesicles with negatively charged phospholipid vesicles and biological membranes. Biochem. 27, 3917.
D. HA-protein induced fusion as a model of SNARE action. HA protein and its mutants can induce fusion, hemifusion, and leakage (which many studies show accompany all forms of fusion): 40 Melikyan et al. (1997) Inner but not outer membrane leaflets control the transition from glycosylphosphatidylinositol-anchored influenza hemagglutinin-induced hemifusion to full fusion. JCellBiol 136, 995 [this is a Wonderful article!]; Earp,... and J. White (2004) The many mechanisms of viral membrane fusion proteins. Curr. Topics Microbiol. Immunol. 285, 25-66; Qiao et al. (1999) A specific point mutant at position 1 of the influenze hemagglutinin fusion peptide displays a hemifusion phenotype. Mol. Biol. Cell 10, 2759; Frolov et al. (2003) Membrane permeability changes at early stages of Influenza hemagglutinin-mediated fusion. Biophys. J. 85, 1725. For an article which uses "the whole toolbox" to analyze how HA-mediated fusion occurs, see Chernomordik et al. (1998) The pathway of membrane fusion catalyzed by influenza hemagglutinin: restriction of lipids, hemifusion, and lipidic fusion pore formation. JCellBiol 140, 1369. A stripped-down model peptide fusogen, and assays of membrane tension--Lau et al. (de Grado) (2004) Oligomerization of fusogenic peptides promotes membrane fusion by enhancing membrane destabilization. Biophys. J. 86, 272-284.
E. Protein associations with membrane lipids, often through lipid head-group specific binding domains: Niggli (2001) Structural properties of lipid-binding sites in cytoskeletal proteins. TIBS 26, 604; Defacque et al. (2002) Phosphoinositides regulate membrane-dependent actin assembly by latex bead phagosomes. Mol. Biol. Cell 13, 1190.
F. Role of specific membrane lipids
1. Sterols slow diffusion from microdomains & modulates phase separation. Valdez-Taubas and Pelham (2003) Slow diffusion of proteins in the yeast plasma membrane allows polarity to be maintained by endocytic cycling. Curr. Biol. 13, 1636; Bacia et al. (2005) Sterol structure determines the separation of phases and the curvature of the liquid-ordered phase in model membranes. PNAS 102, 3272-3277; Samsonov, Mihalyov, and F.S. Cohen (2001) Characterization of cholesterol-sphinomyelin domains and their dynamics in bilayer membranes. Biophys. J. 81, 1486-1500.
2. Roles of diacylglycerol; it induces local curvature and even lamellar (bilayer) to hexagonal phase transition and hence leakage and fusion. Goni and Alonso (1999) Structure and functional properties of diacylglycerols in membranes. Prog. Lipid Res. 38, 1; Siegel et al. (1989) Physiological levels of diacylglycerols in phospholipid membranes induce membrane fusion and stabilize inverted phases. Biochem. 28, 3703; Das and Rand (1984) Diacylglycerol causes major structural transitions in phospholipid bilayer membranes. BBRC 124, 491; Allan et al. (1978) Rapid transbilayer diffusion of 1,2-diacylglycerol and its relevance to control of membrane curvature. Nature 276, 289.
3. Phospholipase C (PLC) can drive fusion by locally producing DAG: 50 Nieva et al. (1989) Liposome fusion catalytically induced by phospholipase C. Biochem. 28, 7364; Burger et al. (1991) Phospholipase C activity-induced fusion of pure lipid model membranes. A freeze-fracture study. BBA 1068, 249; Nieva et al. (1995) Topological properties of two cubic phases of a phospholipid:cholesterol:diacylglycerol aqueous system and their possible implications in the phospholipase C-induced liposome fusion. FEBS Lett 368, 143; Ruiz-Arguello et al. (1998) Vesicle membrane fusion induced by the concerted activities of sphingomyelinase and phospholipase C. JBC 273, 22977.
4. Relationships between the PLC and its DAG product: Nieva et al. (1993) Phospholipase C promoted membrane fusion. Retroinhibition by the end-product diacylglycerol. Biochem. 32, 1054; Basanez et al. (1996) Origin of the lag period in the phospholipase C cleavage of phospholipids in membranes. Concomittant vesicle aggregation and enzyme inactivation. Biochem. 35, 15183; Basanez et al. (1998) Effect of single chain lipids on phospholipase C-promoted vesicle fusion. A test for the stalk hypothesis of membrane fusion. Biochem. 37, 3901.
5. Relationship between Rab and SNARE-driven fusion and PLC/DAG; Jun et al. (2004) Diacylglycerol and its formation by phospholipase C regulate Rab and SNARE-dependent yeast vacuole fusion. JBC 279, 53186.
6. Effects of "cone" and "wedge" lipids: Rigoni et al. (2005) Equivalent effects of snake PLA2 neurotoxins and lysophospholipid-fatty acid mixtures. Science 310, 1678-1680; Hughes et al. (2004) Phospholipase D1 regulates secretagogue-stimulated insulin release in pancreatic b-cells. JBC 279, 27534-41.
7. Interrelationship among curvature and specific lipids--Roux et al. (B. Goud) (2005) Role of curvature and phase transition in lipid sorting and fission of membrane tubules. EMBO J. 24, 1537-1545.
8. Phosphoinositides form separate phases--Redfern and Gericke (2004) Domain formation in phosphatidylinositol monophosphate/phosphatidylcholine mixed vesicles. Biophys. J. 86, 2980-2992.
Fusion via a hemifusion intermediate. Review--Chernomordik & Kozlov (2005) Membrane hemifusion: crossing a chasm in two leaps. Cell 123, 375-382. Xu et al. (2005) Hemifusion in SNARE-mediated membrane fusion. Nature Struct & Mol Biol. 12, 417-422; Lu et al. (2005) Membrane fusion induced by neuronal SNAREs transits through hemifusion. J. Biol. Chem. 280, 30538-41. Reese et al. (2005) Trans-SNARE pairing can precede a hemifusion intermediate in intracellular membrane fusion. Nature 436, 410-414; Reese and Mayer (2005) Transition from hemifusion to pore opening is rate limiting for vacuole membrane fusion. J. Cell Biol. 171, 981-90.
For common factors of membrane fusion pathways. For a general review, see Jahn and Sudhof (1999) Membrane fusion and Exocytosis. Ann Rev. Biochem. 68, 863.
II. SNAREs, NSF (Sec18p), SNAP (Sec17p), and Sec1; for reviews, see Pfeffer (1996) Transport vesicle docking; SNAREs and associates. Ann. Rev. Cell Biol. 12, 441; Gerst (1999) Cell. Mol .Life Sci. 55, 707; 60 *Rizo and Sudhof (2002) SNAREs and MUNC18 in synaptic vesicle fusion. Nature Reviews Neuroscience 3, 641; Ungar and Hughson (2003) SNARE protein structure and function. Ann Rev Cell Dev Biol 19, 493-517; Jahn & Scheller (2006) SNAREs- engines for membrane fusion. Nature Rev. Mol. Cell Biol. 7, 631-43.
1. Discovery, as neural proteins (Bennett et al. (1992) Syntaxin: A synaptic protein implicated in docking of synaptic vesicles at presynaptic active zones. Science 257, 255) and as SNAP receptor (Sollner et al. (1993) SNAP receptors implicated in vesicle targeting and fusion. Nature 362, 318.)
2. Biochemistry of pure, recombinant proteins: Pevsner et al. (1994) Specificity and regulation of a synaptic vesicle docking complex. Neuron 13, 353; Fasshauer et al. (1997) Structural changes are associated with soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor complex formation. JBC 272, 28036; Hanson et al. (1995) The N-ethylmaleimide-sensitive fusion protein and a-SNAP induce a conformational change in syntaxin. JBC 270, 16955.
2.5 N-domains--Tochio et al. (2001) An autoinhibitory mechanism for nonsyntaxin SNARE proteins revealed by the structure of Ykt6p. Science 293, 698-702.
3. Structure of the SNARE complex, and the 20S complex with NSF and SNAP: Yu et al. (1999) NSF N-terminal domain crystal structure: Models of NSF function. Mol. Cell 4, 97; Sutton et al (1998) Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4A resolution. Nature 395, 347; Rice and Brunger (1999) Crystal structure of the vesicular transport protein Sec17: Implications for SNAP function in SNARE complex disassembly. Mol Cell 4, 85; and, for a classification of SNAREs as Qa-c, R, see Fukuda et al. (2000) Functional architecture of an intracellular membrane t-SNARE. Nature 407, 198-202.
3.5 Role of the 0-layer: 3Q1R is important, and position of these residues matters--Graf et al. (Jahn) (2005) Identification of functionally interacting SNAREs by using complementary substitutions in the conserved "0" layer. Mol Biol Cell 16, 2263-2274. For NSF/SNAP action--Lauer et al (2006) SNARE complex zero layer residues are not critical for N-ethylmaleimide-sensitive factor-mediated disassembly. JBC 281, 14823-32.
3.75 Synaptobrevin TM domain "tilt" destabilizes membranes for fusion--Bowen & Brunger (2006) Conformation of the synaptobrevin transmembrane domain. PNAS 103, 8378-8383.
4. NSF/SNAP function. Hanson et al. (1997) Structure and conformational changes in NSF and its membrane receptor complexes visualized by quick freeze/deep-etch electron microscopy. Cell 90, 523. NSF can have additional functions (Muller et al. (1999) An NSF function distinct from ATPase-dependent SNARE disassembly is essential for Golgi membrane fusion. Nature Cell Biol. 1, 335; Muller et al. (2002) Sequential SNARE disassembly and GATE-16-GOS28 complex assembly mediated by distinct NSF activities drives Golgi membrane fusion. J. Cell Biol. 157, 1161). For a real mind-bender, try to explain the observations in Gerrard et al. (2000) The yeast endosomal t-SNARE, Pep12p, functions in the absence of its transmembrane domain. Traffic 1, 45.
5. Assays of cis and trans SNARE complexes: Ungermann et al. (1998) A vacuolar v-t-SNARE complex, the predominant form in vivo and on isolated vacuoles, is disassembled and activated for docking and fusion. JCB140, 61; Otto et al. (1997) Assembly and disassembly of a ternary complex of synaptobrevin, syntaxin, and SNAP-25 in the membrane of synaptic vesicles. PNAS 94, 6197.
6. Specificity of SNARE pairing: SNARE complex formation is promiscous, either with recombinant proteins (Yang et al. (1999) SNARE interactions are not selective. JBC 274, 5649) or in vivo (Grote and Novick (1999) Promiscuity in Rab-SNARE interactions. Mol. Biol. Cell 10, 4149; Holthuis et al. (1997) Two syntaxin homologues in the TGN/endosomal system of yeast. EMBO J. 16, 113.) For data showing a v-SNARE can mediate both forward and reverse traffick, see Lewis et al. (1997) A novel SNARE complex implicated in vesicle fusion with the endoplasmic reticulum. EMBO J. 16, 3017, and also Fischer von Mollard et al. (1997) The yeast v-SNARE Vti1p mediates two vesicle transport pathways through interactions with the tSNAREs Sed5p and Pep12p.
7. Other SNARE associations:
a. with Sec1: Yang et al. (2000) nSec1 binds a closed conformation of syntaxin 1A. JCB 148, 247; Misura et al. (2000) Three-dimensional structure of the neuronal Sec1-Syntaxin 1a complex. Nature 404, 355. For the heretical view that Sec1 binds to the SNARE complex, rather than the isolated t-SNARE, see 70 Carr et al. (1999) Sec1p binds to SNARE complexes and concentrates at sites of secretion. JCB 146, 333. This is supported by the recent Fisher et al. (2001) Control of fusion pore dynamics during exocytosis by Munc18. Science 291, 875. Do SM proteins confer SNARE specificity? Peng and Gallwitz (2002) Sly1 protein bound to Golgi syntaxin Sec5p allows assembly and contributes to specificity of SNARE fusion complexes. J. Cell Biol. 157, 645; Bryant and James (2001) Vps45p stabilizes the syntaxin homologue Tlg2p and positively regulates SNARE complex formation. EMBO J. 20, 3380. Though SM proteins may not be compartment or SNARE specific (Peng and Gallwitz (2004) Multiple SNARE interactions of an SM protein: Sec5p/Sly1p binding is dispensable for transport. EMBO J. 23, 3939), they do participate in the SNARE cycle (Bryant and James (2003) The Sec1p/Munc18p protein Vps45p cycles on and off membranes during vesicle transport. JCell Biol. 161, 691) and can directly stimulate the reconstituted SNARE-driven liposome fusion (Scott et al. (2004) Sec1p directly stimulates SNARE-mediated membrane fusion in vitro. JCellBiol 167, 75). Sec1 acts downstream of trans-SNARE assembly: Grote et al. (2000) Ordering of the final events in yeast exocytosis. J. Cell Biol. 151, 439-51. Might SM proteins act other than via syntaxin?--Ciufo et al. (2005) Munc 18-1 regulates early and late stages of exocytosis via syntaxin-independent protein interactions. Mol. Biol. Cell 16, 470-482. SM/SNARE binding modes--Carpp et al. (2006) The Sec1p/Munc18 protein Vps45 binds its cognate SNARE proteins via two distinct modes. J. Cell Biol. 173, 927-936.
b. with Ca channels: See Bennett et al. (1992), section 1 above.
c. with Vac1p and EEA1; Shorter et al. (2002) Sequential tethering of Golgins and catalysis of SNAREpin assembly by the vesicle-tethering protein p115. J. Cell Biol. 157, 45-62. "Mint" proteins are Munc-interacting proteins, such as Vac1p, SSO2p, and Mso1p--Knop et al. (2005) Molecular interactions position Mso1p, a novel PTB domain homologue, in the interface of the exocyst complex and the exocytic SNARE machinery in yeast. Mol Biol Cell 16, 4543-56.
d. with each other- Tokumaru et al. (2001) SNARE complex oligomerization by spaphin/complexin is essential for synaptic vesicle exocytosis. Cell 104, 421.
e. with tomosyn: Hatsuzawa et al. (2003) The R-SNARE motif of tomosyn forms SNARE core complexes with syntaxin 1 and SNAP-25 and down-regulates exocytosis. JBC 278, 31159.
f. With Hrs: 80 Yan et al. (2004) Ca and N-Ethylmaleimide-sensitive factor differentially regulate disassembly of SNARE complexes on early endosomes. JBC 279, 18270.
g. with lipid; synaptobrevins like Nyv1p have trp residues that directly insert into lipid and modulate trans-SNARE pairing; Ca2+/calmodulin may regulate their accessibility! Quetglas et al. (2002) Calmodulin and lipid binding to synaptobrevin regulates calcium-dependent exocytosis. EMBO J. 21, 3970; Kweon et al. (2003) Regulation of neuronal SNARE assembly by the membrane. Nat Struct. Biol. 10, 440-447; Chen et al. (2004) Constitutive versus regulated SNARE assembly: a structural basis. EMBO J. 23, 681; deHaro et al. (2004) Ca/calmodulin transfers the membrane-proximal lipid-binding domain of the v-SNARE synaptobrevin from cis to trans bilayers. PNAS 101, 1578. Arachidonic acid regulates SNARE pairing, in vivo or with pure proteins--Rickman & Davletov (2005) Arachidonic acid allows SNARE complex formation in the presence of Munc18. Chem & Biol 12, 545-553; Darios & Davletov (2006) Omega-3 and omega-6 fatty acids stimulate cell membrane expansion by acting on syntaxin 3. Nature 440, 813-817.
h. with rafts: Chamberlain et al. (2001) SNARE proteins are highly enriched in lipid rafts in PC12 cells: Implications for the spatial control of exocytosis. PNAS 98, 5619.
i. with Cdc42p; Nevins and Thurmond (2005) A direct interaction between Cdc42 and VAMP2 regulates SNARE-dependent insulin exocytosis. J. Biol. Chem. 280, 1944-1952.
j. With complexins and synaptotagmin: Tang et al. (Sudhof) (2006) A complexin/synaptotagmin 1 switch controls fast synaptic vesicle exocytosis. Cell 126, 1175-1187; Rickman & Davletov (2003) Mechanisms of calcium-independent synaptotagmin binding to target SNAREs. JBC 278, 5501-4; Hu et al. (Davletov) (2002) Action of complexin on SNARE complex. JBC 277, 41652-6. SNAREs with synaptotagmin fuse much, much better and without lysis--Bhalla et al. (Chapman) (2006) Ca2+-synaptotagmin directly regulates t-SNARE function during reconstituted membrane fusion. Nature Struct & Mol Biol 13, 323-330.
k. With dynamin (Vrp1p)--Peters et al. (2004) Mutual control of membrane fission and fusion proteins. Cell 119, 667-678.
8. Modulation of function by phosphorylation: Cabaniols et al. (1999) Phosphorylation of SNAP-23 by the novel kinase SNAK regulates t-SNARE complex assembly. Mol. Biol. Cell 10, 4033; Hirling and Scheller (1996) Phosphorylation of synaptic vesicle proteins: Modulation of the a-SNAP interaction with the core complex. PNAS 93, 11945; Fujita et al. (1996) Phosphorylation of Munc-18/nSec1/rbSec1 by protein kinase C. JBC271, 7265; Marash and Gerst (2001) t-SNARE dephosphorylation promotes SNARE assembly and exocytosis in yeast. EMBO J. 20, 411. The last of these makes the striking finding that phosphorylation seems to govern the specificity of SNARE pairing.
9. Models of SNARE function:
a. SNAREpins drive fusion: 90 Skehel and Wiley (1998) Coiled coils in both intracellular vesicle and viral membrane fusion. Cell 95, 871; Weber et al. (1998) SNAREpins: Minimal machinery for membrane fusion. Cell 92, 759; Weber et al. (2000) SNAREpins are functionally resistant to disruption by NSF and SNAP. JCB 149, 1063; however, see Lonart and Sudhof (2000) Assembly of SNARE core complexes prior to neurotransmitter release sets the readily releasable pool of synaptic vesicles. JBC 275, 27703. McNew et al (2000) Compartmental specificity of cellular membrane fusion encoded in SNARE proteins. Nature 407, 153; however, see Scales et al. (2000) The specifics of membrane fusion, Nature 407, 144, and Coorsen et al. (2003) Regulated secretion: SNARE density, vesicle fusion, and calcium dependence. J. Cell Sci. 116, 2087. Questions of the relevance of this specificity in liposomes to biol. membrane fusion--Brandhorst et al (Jahn) (2006) Homotypic fusion of early endosomes: SNAREs do not determine fusion specificity. PNAS 103, 2701-6. Other liposomal models: 1. NSF/SNAP-driven fusion; Otter-Nilsson et al (1999) Cytosolic ATPases, p97 and NSF, are sufficient to mediate rapid membrane fusion. EMBO J. 18, 2074. 2. Clathrin-driven fusion; Maezawa and Yoshimura (1990) Assembly of clathrin molecules on liposomal membranes: A possible event necessary for induction of membrane fusion. BBRC 173, 134. And, for a warning about the liposome fusion assay, see Duzgunes et al. (1987) Lipid mixing during membrane aggregation and fusion; Why fusion assays disagree. Biochem. 26, 8435. Very important recent work strengthens this model: 100 Hu et al. (2003) Fusion of cells by flipped SNAREs. Science 300, 1745; Fix et al. (2004) Imaging single membrane fusion events mediated by SNARE proteins. PNAS 101, 7311; and, a particularly convincing study is *Tucker et al. (2004) Reconstitution of Ca-regulated membrane fusion by synaptotagmin and SNAREs. Science 304, 435. For a biophysical view of the structures, see Cho et al. (2002) SNAREs in opposing bilayers interact in a circular array to form conducting pores. Biophys. J. 83, 2522. Snarepins form long before fusion: Zhang et al. (2004) SNARE assembly and membrane fusion, a kinetic analysis. J. Biol. Chem. 279, 38668-92; Melia et al. (2006) Lipidic antagonists to SNARE-mediated fusion. J. Biol. Chem. 281, 29597-605. Is there lipid specificity?--Vicogne et al. (2006) Asymmetric phospholipid distribution drives in vitro reconstituted SNARE-dependent membrane fusion. PNAS 103, 14761-6.
Are liposomes at high SNARE concentrations merely undergoing lysis/reannealing? Dennison et al. (Lentz, Brunger) (2006) Neuronal SNAREs do not trigger fusion between synthetic membranes but do promote PEG-mediated membrane fusion. Biophys. J. 90, 1661-1675; Chen et al. (2006) SNARE-mediated lipid mixing depends on the physical state of the vesicles. Biophys. J. 90, 2062-2074.
SNAREs with synaptotagmin fuse much, much better and without lysis--Bhalla et al. (Chapman) (2006) Ca2+-synaptotagmin directly regulates t-SNARE function during reconstituted membrane fusion. Nature Struct & Mol Biol 13, 323-330.
b. SNAREs are only needed until docking is complete: Ungermann et al. (1998) Defining the functions of trans-SNARE pairs. Nature 396, 543; Tahara et al. (1998) Calcium can disrupt the SNARE protein complex on sea urchin egg secretory vesicles without irreversibly blocking fusion. JBC 273, 33667; Coorssen et al. (1998) Biochemical and functional studies of cortical vesicle fusion: The SNARE complex and Ca2+ sensitivity. J. Cell Biol. 143, 1845; Broadie et al. (1995) Syntaxin and synaptobrevin function downstream of vesicle docking in Drosophila. Neuron 15, 663.
c. Syntaxin lining a fusion pore--Han et al. (Chapman) (2004) Transmembrane segments of syntaxin line the fusion pore of Ca2+-triggered exocytosis. Science 304, 289-292.
d. The tSNARE functions with V0; *Peters et al. (2001) Trans-complex formation by proteolipid channels in the terminal phase of membrane fusion. Nature 409, 581. In this context, see Galli et al. (1996) The V0 sector of the V-ATPase, synaptobrevin, and synaptophysin are associated on synaptic vesicles in a Triton X100-resistant, freeze-thawing sensitive complex. JBC 271, 2193; also, David et al. (1998) Involvement of long-chain fatty acid elongation in the trafficking of secretory vesicles in yeast. JCB143, 1167; this paper shows that t-SNAREs can be essential but v-SNAREs (for yeast exocytosis) can be bypassed. Vo association with actin--Vitavska et al. (2003) A novel role for subunit C in mediating binding of the H+-V-ATPase to the actin cytoskeleton. J. Biol. Chem. 278, 18499-505; Sweet!--confirmation from another systems-- Liegeois et al. (2006) The Vo-ATPase mediates apical secretion of exosomes containing Hedgehog-related proteins in Caenorhabditis elegans. J. Cell Biol. 173, 949-961; Galli et al. (de Camilli) (1996) The Vo sector of the V-ATPase, synaptobrevin, and synaptophysin are associated on synaptic vesicles in a Triton X-100-resistant, freeze-thawing sensitive, complex. J. Biol. Chem. 271, 2193-8; Hiesinger et al. (2005) The Vo-ATPase subunit a1 is required for a late step in synaptic vesicle exocytosis in Drosophila. Cell 121, 607-620.
III. GTPases, their effectors, and tethering complexes; for reviews, see 110 *Chavrier and Goud (1999) The role of ARF and Rab GTPases in membrane transport. Curr Op Cell Biol. 11, 466; Novick and Zerial (1997) The diversity of Rab proteins in vesicle transport. Curr Op Cell Biol. 9, 496; Schimmoller et al. (1998) Rab GTPases, directors of vesicle docking JBC 273, 22161; Waters and Hughson (2000) Membrane tethering and fusion in the secretory and endocytic pathways. Traffic 1, 588.
1. Novick's discovery of Ypt/Rab function; Sec4p. *Goud et al. (1988) A GTP-binding protein required for secretion rapidly associates with secretory vesicles and the plasma membrane in yeast. Cell 53, 753; however, raising questions about these proteins as the primary agent of organelle identity, see Brennwald and Novick (1993) Interactions of three domains distinguishing the Ras-related GTP-binding proteins Ypt1p and Sec4. Nature 362, 560.
2. Basic cycle between GDP and GTP forms. Walworth et al. (1989) Mutational analysis of SEC4 suggests a cyclical mechanism for the regulation of vesicular traffic. EMBO J. 8, 1685; Collins et al. (1997) Interactions of nucleotide release factor Dss4p with sec4p in the post-Golgi secretory pathway of yeast. JBC 272, 18281.
3. Structures of Rab:GDP, GTP, and with effectors. Minireview: Gonzalez and Scheller (1999) Regulation of membrane trafficking; Structural insights from a Rab/effector complex. Cell 96, 755.
4. Rab GTPases can act in tandem: Oritz et al. (2002) Ypt32 recruits the Sec4p guanine nucleotide exchange factor, Sec2p, to secretory vesicles; evidence for a Rab cascade in yeast. J. Cell Biol. 157, 1005.
Heavily-studied "Rab Effectors"
5. Exocyst. For a minireview, see 120 Finger and Novick (1998) Spatial regulation of exocytosis. J. Cell Biol. 142, 609. Primary ref's: TerBush et al. (1996) The exocyst is a multiprotein complex required for exocytosis in Saccharomyces cerevisiae. EMBO J. 15, 6483; Guo et al. (1999) The exocyst is an effector for Sec4p, targeting secretory vesicles to sites of exocytosis. EMBO J. 18, 1071.
6. Uso1/p115/TRAPP. Cao et al. (1998) Initial docking of ER-derived vesicles requires Uso1p and Ypt1p but is independent of SNARE proteins. EMBO J. 17, 2156; Shorter et al. (2002) Sequential tethering of Golgins and catalysis of SNAREpin assembly by the vesicle-tethering protein p115. J. Cell Biol. 157, 45; Beard,...and G. Warren (2005) A cryptic Rab1-binding site in the p115 tethering protein. J. Biol. Chem. 280, 25840-25848; Sacher et al. (1998) TRAPP, a highly conserved novel complex on the cis-Golgi that mediates vesicle docking and fusion. EMBO J. 17, 2494. For a third ER to Golgi tethering complex, see VanRheenan et al. (1999) Sec34p, a protein required for vesicle tethering to the yeast Golgi apparatus, is in a complex with Sec35p. J. Cell Biol. 147, 729.
7. Mammalian endosome (Rab5). Woodman (2000) Biogenesis of the sorting endosome: The role of Rab5. Traffic 1, 695. Stenmark et al. (1994) Inhibition of Rab5 GTPase activity stimulates membrane fusion in endocytosis. EMBO J. 13, 1287; Stenmark et al. (1995) Rabaptin-5 is a direct effector of the small GTPase Rab5 in endocytic membrane fusion. Cell 83, 423; *Horiuchi et al. (1997) A novel Rab5 GDP/GTP exchange factor complexed to rabaptin-5 links nucleotide exchange to effector recruitment and function. Cell 90, 1149; Simonsen et al. (1998) EEA1 links PI(3)K function to rab5 regulation of endosome fusion. Nature 394, 494; Christoforidis et al. (1999) The Rab5 effector EEA1 is a core component of endosome docking Nature 397, 621; 130 Christoforidis et al. (1999) Phosphatidylinositol-3-OH kinases are Rab5 effectors. Nature Cell Biol. 1, 249. For how the proteins work together, DeRenzis et al. (2002) Divalent Rab effectors regulate the subcompartmental organization and sorting of early endosomes. Nature Cell Biol. . For a review, see Miaczynska and Zerial (2002) Mosaic organization of the endocytic pathway. Exp. Cell Res. 272, 8.
8. Yeast endosome (Vps21). Burd et al. (1997) A novel Sec18p/NSF-dependent complex required for Golgi to endosome transport in yeast. Mol Biol Cell 8, 1089; Tall et al. (1999) The phosphatidylinositol 3-phosphate binding protein Vac1p interacts with a Rab GTPase and a Sec11p homologue to facilitate vesicle-mediated vacuolar protein sorting. Mol. Biol. Cell 10, 1873.
9. HOPS is of interest because this Ypt/Rab effector complex contains the Sec1 homolog (Vps33p) and the nucleotide exchange factor (Vps39p) and yet appears to interact with SNAREs prior to engaging Ypt7p; Price et al. (2000) The docking stage of yeast vacuole fusion requires the transfer of proteins from a cis-SNARE complex to a Rab/Ypt protein. JCB148, 1231; Seals et al. (ref in IF3 above); Stroupe et al. (2006) Purification of active HOPS complex reveals its affinities for phosphoinositides and the SNARE Vam7p. EMBO J. 25, 1579-1589; regulation of vacuole fusion by phosphorylation of HOPS is presented in Tracy et al. (2005) The vacuolar kinase Yck3 maintains organelle fragmentation by regulating the HOPS tethering complex. HOPS interacts with actin & tubulin: Richardson et al. (2004) Mammalian late vacuole protein sorting orthologues participate in early endosomal fusion and interact with the cyotskeleton. Mol Biol Cell 15, 1194-1210.
10. Rabphilin binds Rabs, lipids (via its C2B domain), and the SNAP-25 SNARE--Tsuboi & Fukuda (2005) The C2B domain of rabphilin directly interacts with SNAP-25 and regulates the docking step of dense core vesicle exocytosis in PC12 cells. JBC 280, 39253-59.
11. VFT complex at the late Golgi; Siniossoglou & Pelham (2002) Vps51p links the VFT complex to the SNARE Tlg1p. J. Biol. Chem. 277, 48318.
Rho-family GTPases also function in trafficking
Doussau et al. (2000) A Rho-related GTPase is involved in Ca2+-dependent neurotransmitter exocytosis. J. Biol. Chem. 275,7764; Guo et al. (2001) Spatial regulation of the exocyst complex by Rho1 GTPase. Nature Cell Biol. 3, 353; Zhang et al. (2001) Cdc42 interacts with the exocyst and regulates polarized secretion. J. Biol. Chem. 276, 46745; 140 Adamo et al. (1999) The Rho GTPase Rho3 has a direct role in exocytosis that is distinct from its role in actin polarity. Mol. Biol. Cell 10, 4121; Adamo et al. (2001) Yeast Cdc42 functions at a late step in endocytosis, specifically during polarized growth of the emerging bud. J. Cell Biol. 155, 581.
IV. Calcium, phosphoinositides, and actin
A. Calcium transporters, sensors, and effectors. For a good review, see *Bennett (1997) Ca and the regulation of neurotransmitter secretion. Curr Op Neurobiol. 7, 316. Also Burgoyne and Morgan (1998) Calcium sensors in regulated exocytosis. Cell Calcium 24, 367. Most current is Taylor (2002) Controlling calcium entry. Cell 111, 767.
1. Synaptotagmin as calcium sensor; Desai et al. (2000) The C2B domain of synaptotagmin is a Ca-sensing module essential for exocytosis. JCB 150, 1125. Fernandez-Chacon et al. (2001) Synaptotagmin I functions as a calcium regulator of release probability. Nature 410, 41. Zhang et al. (2002) Ca2+-dependent synaptotagmin binding to SNAP-25 is essential for Ca2+-triggered exocytosis. Neuron 34, 599; Bai et al. (2000) Membrane-embedded synaptotagmin penetrates cis or trans target membranes and clusters via a novel mechanism. J. Biol. Chem. 275, 25427; Sudhof (2002) Synaptotagmins: Why so many? J. Biol. Chem. 277, 7629. Most recently, an impressive direct demonstration that Ca:synaptotagmin can cooperate with SNAREs to mediate bilayer fusion: *Tucker et al. (2004) Reconstitution of Ca-regulated membrane fusion by synaptotagmin and SNAREs. Science 304, 289. Might a major role of SNAREs be to keep synaptotagmin at the right place?--see Zimmerberg et al. (2006) Synaptotagmin: fusogenic role for calcium sensor? Nature Struct & Mol Biol. 13, 301-313.
2. Calmodulin as the primary Ca sensor: Garofalo et al. (1983) Calmodulin antagonists inhibit secretion in paramecium. JCB 96, 1072; 150 Kenigsberg and Trifaro (1985) Microinjection of calmodulin antibodies into cultured chromaffin cells blocks catecholamine release in response to stimulation. Neuroscience 14, 335-347; Brockerhoff and Davis (1992) Calmodulin concentrates at regions of cell growth in Saccharomyces cerevisiae. JCB 118, 619; Kubler et al. (1994) Calcium-independent calmodulin requirement for endocytosis in yeast. EMBO J. 13, 5539; Colombo et al. (1997) Calmodulin regulates endosome fusion. JBC 272, 7707; Peters and Mayer (1998) Ca/calmodulin signals the completion of docking and triggers a late step of vacuole fusion. Nature 396, 575; O'Connor et al. (1999) Calmodulin dependence of presynaptic metabotropic glutamate receptor signaling. Science 286, 1180; Peracchia et al. (2000) Calmodulin directly gates gap junction channels. JBC 275, 262204; Quetglas et al. (2002) Calmodulin and lipid binding to synaptobrevin regulates calcium dependent exocytosis. EMBO J. X, 3970; Quetglas et al. (2000) Ca2+-dependent regulation of synaptic SNARE complex assembly via a calmodulin and phospholipid-binding domain of synaptobrevin. Proc. Natl. Acad. Sci. USA 97, 9565. Calmodulin is often an ion-channel subunit; Saimi and Kung (2002) Calmodulin as an ion channel subunit. Ann. Rev. Physiol. 64, 289.
3. Syncollin: 160 Edwardson et al. (1997) The secretory granule protein syncollin binds to syntaxin in a Ca-sensitive manner. Cell 90, 325.
4. Syntaxin/Ca channel interactions: Bezprozvanny et al. (1995) Functional impact of syntaxin on gating of N-type and Q-type calcium channels. Nature 378, 623; Sheng et al. (1996) Calcium-dependent interaction of N-type calcium channels with the synaptic core complex. Nature 379, 451; Wiser et al. (1996) Functional interaction of syntaxin and SNAP-25 with voltage-sensitive L- and N-type Ca channels. EMBO J. 15, 4100; Jarvis et al. (2002) Molecular determinants of syntaxin 1 modulation of N-type calcium channels. J. Biol. Chem. 277, 44399.
5. Annexin; Faure et al. (2002) Annexin 2 "secretion" accompanying exocytosis of chromaffin cells: Possible mechanisms of annexin release. Exp. Cell Res. 276, 79.
6. Ca has multiple roles (in priming, as well as triggering)--Rettig and Neher (2002) Emerging roles of presynaptic proteins in Ca-triggered exocytosis. Science 298, 781-5.
B. Phosphatidylinositol phosphatides and downstream elements. For review, see Huijbregts et al. (2000) Lipid metabolism and regulation of membrane trafficking. Traffic 1, 195; De Camilli et al. (1996) Phosphoinositides as regulators in membrane traffic. Science 271, 1533; Wurmser et al. (1999) Phosphoinositide 3-kinases and their FYVE domain-containing effectors as regulators of vacuolar/lysosomal membrane trafficking pathways. JBC 274, 9129. Huijbregts et al. (2000) Lipid metabolism and regulation of membrane trafficking. Traffic 1, 195; Osborne et al. (2001) Phosphoinositides as key regulators of synaptic function. Neuron 32, 9; McLaughlin et al. (2002) PIP2 and proteins: Interactions, organization, and information flow. Ann. Rev. Biophys. Biomol. Struct. 31, 151-175.
1. The Emr story: A protein kinase (Vps15p) binds a PI-3-kinase (Vps34p) to the Golgi; 170 Schu et al. (1993) Phosphatidylinositol 3-kinase encoded by yeast VPS34 gene essential for protein sorting. Science 260, 88. Fab1p converts PI(3)P to PI(3,5)P2; Gary et al. (1998) Fab1p is essential for PtdIns(3)P-5-kinase activity and the maintenance of vacuolar size and membrane homeostasis. JCB 143, 65 . This lipid binds proteins of trafficking with FYVE domains; Burd and Emr (1998) Phosphatidylinositol (3)-phosphate signaling mediated by specific binding to RING FYVE domains. Mol. Cell 2, 157 . It is itself degraded by delivery into the endosome by invagination and delivery to the vacuole and degradation; Wurmser and Emr (1998) Phosphoinositide signaling and turnover: PtdIns(3)P, a regulator of membrane traffic, is transported to the vacuole and degraded by a process that requires lumenal vacuolar hydrolase activities. EMBO J. 17, 4930.
2. The phospholipase D cycle; it makes PA + IP2 from PIP3. PA activates PI(4P)-5-kinase to make more PIP3; thus, a positive feedback loop, making a highly acidic patch. Liscovitch et al. (1994) Novel function of phosphatidylinositol 4,5-bisphosphate as a cofactor for brain membrane phospholipasse D. JBC 269, 21403. The ARF GTPase activates this process (Godi et al. (1999) ARF mediates recruitment of PtdIns-4-OH kinase and stimulates synthesis of PtdIns(4,5)P2 on the Golgi complex. Nature Cell Biol. 1, 280) and PI(4,5)P2 binds proteins via their Plextrin homology (PH) domains. Actin is induced to polymerize at these acidic lipid domains; Shibasaki et al. (1997) Massive actin polymerization induced by phosphatidylinositol-4-phosphate -5kinase in vivo. JBC 272, 7578; Lin et al. (1997) Gelsolin binding to phosphatidylinositol 4,5-bisphosphate is modulated by calcium and pH. JBC 272, 20443. Gelsolin is an actin capping protein which promotes actin polymerization.
3. Phospholipase C, the generation of IP3 and diacylglycerol, and IP3-mediated Ca flux ; Sullivan et al. (1993) Cell 73, 1411. However, see Patterson et al. (2002) Phospholipase C-g is required for agonist-induced Ca2+ entry. Cell 111, 529. In the latter paper they show that the active site of phospholipase C isn't needed for it to support Ca flux, though its SH3 domain is required.
4. Fitting the lipid and protein pieces together: 180 Chung et al. (1998) The C2 domains of rabphilin 3A specifically bind phosphatidylinositol 4,5-bisphosphate containing vesicles in a Ca2+ dependent manner. J. Biol. Chem. 273, 10240.
5. Rab regulation of phosphoinositides: Shin et al. (incl. deCamilli and Zerial) (2005) An enzymatic cascade of Rab5 effectors regulates phosphoinositide turnover in the endocytic pathway. J. Cell Biol. 170, 607-618.
6. Phosphoinositides and Vam7p: Boeddinghaus et al. (2002) A cycle of Vam7p release from and PtdIns3-P-dependent rebinding to the yeast vacuole is required for homotypic vacuole fusion. J. Cell Biol. 157, 79-89.
C. Actin regulates membrane fusion events, though there is little distinguishing its role in promoting the movement of vesicles to their docking sites from whether they promote fusion once docking has occurred. Jahraus et al. (2001) ATP-dependent membrane assembly of F-actin facilitates membrane fusion. Mol. Biol. Cell 12, 155; Jahraus et al. (2004) Fusion between phagosomes, early and late endosomes: A role for actin in fusion between late, but not early, endocytic organelles. Mol. Biol. Cell 15, 345; Gasman et al. (2004) Regulated exocytosis in neuroendocrine cells: A role for subplasmalemmal Cdc42/N-WASP-induced actin filaments. Mol. Biol. Cell 15, 520; Eitzen et al. (2002) Remodeling of organelle-bound actin is required for yeast vacuole fusion. JCellBiol. 158, 669; McKane et al. (2005) A mammalian actin substitution in yeast actin (H372R) causes a suppressible mitochondria/vacuole phenotype. J. Biol. Chem. 280, 36494-501. Actin on vacuoles: Drengk et al. (2003) A coat of filamentous actin prevents clustering of late-endosomal vacuoles in vivo. Curr. Biol. 13, 1814-1819.
Dynamin directly binds to PI(4,5)P2, to actin and actin-regulatory proteins, and to SNAREs; it could play a key central role. Orth and McNiven (2003) Dynamin at the actin-membrane interface. CurrOpCellBiol 15, 31; Yu and Cai (2004) The yeast dynamin-related GTPase Vps1p functions in the organization of the actin cytoskeleton via interactions with Sla1p. JCellSci 117, 3839; Peters et al. (2004) Mutual control of membrane fission and fusion proteins. Cell 119, 667; Antonny (2004) SNARE filtering by dynamin. Cell 119, 581.
Vo binds actin: Vitavska et al. (2005) The V-ATPase subunit C binds to polymeric F-actin as well as to monomeric G-actin and induces cross-linking of actin filaments. J. Biol. Chem. 280, 1070-1076.
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