Developmental Cell Article The CORVET Tethering Complex Interacts with the Yeast Rab5 Homolog Vps21 and Is Involved in Endo-Lysosomal Biogenesis Karolina Peplowska, 1 Daniel F. Markgraf, 1,3 Clemens W. Ostrowicz, 1,3 Gert Bange, 2 and Christian Ungermann 1, * 1 University of Osnabru ¨ ck, Department of Biology, Biochemistry Section, Barbarastrasse 13, 49076 Osnabru ¨ ck, Germany 2 Biochemie Zentrum der Universita ¨ t Heidelberg (BZH), Im Neuenheimer Feld 328, 69120 Heidelberg, Germany 3 These authors contributed equally to this work. *Correspondence: [email protected]DOI 10.1016/j.devcel.2007.03.006 SUMMARY The dynamic equilibrium between vesicle fis- sion and fusion at Golgi, endosome, and vacu- ole/lysosome is critical for the maintenance of organelle identity. It depends, among others, on Rab GTPases and tethering factors, whose function and regulation are still unclear. We now show that transport among Golgi, endo- some, and vacuole is controlled by two homol- ogous tethering complexes, the previously identified HOPS complex at the vacuole and a novel endosomal tethering (CORVET) com- plex, which interacts with the Rab GTPase Vps21. Both complexes share the four class C Vps proteins: Vps11, Vps16, Vps18, and Vps33. The HOPS complex, in addition, con- tains Vps41/Vam2 and Vam6, whereas the CORVET complex has the Vps41 homolog Vps8 and the (h)Vam6 homolog Vps3. Strikingly, the CORVET and HOPS complexe can intercon- vert; we identify two additional intermediate complexes, both consisting of the class C core bound to Vam6-Vps8 or Vps3-Vps41. Our data suggest that modular assembled tethering complexes define organelle biogenesis in the endocytic pathway. INTRODUCTION Vesicle-mediated protein transport between organelles of the endomembrane system depends on a dynamic equi- librium between fission and fusion. Alterations of this bal- ance lead to a loss of organelle identity and subsequently to disease (Di Pietro and Dell’Angelica, 2005; Munro, 2004). Vesicle fusion requires Rab GTPase-dependent tethering of the vesicle, followed by the fusion process, which is driven by SNARE proteins residing on the vesicle and organelle membrane. Rab GTPases have been impli- cated as key regulators of fusion (Grosshans et al., 2006). To be able to bind their effectors, Rab GTPases need to be converted from the inactive GDP to the active GTP form. The conversion depends on guanine nucleotide exchange factors (GEFs), which vary in size and domain compo- sition. Inactivation of Rab proteins depends on Rab GTPase- activating proteins (GAPs), which trigger GTP hydrolysis (Haas et al., 2005). Tethering factors or large multimeric tethering com- plexes (tethers) cooperate with Rab GTPases to capture vesicles and trap them prior to the action of SNAREs (Beh- nia and Munro, 2005; Grosshans et al., 2006). Tethers are therefore thought to coordinate Rab and SNARE function and provide an essential layer of specificity to fusion reac- tions. Several large tethering complexes have been iden- tified, including the exocyst at the plasma membrane, the TRAPP and the COG complex at the Golgi, the GARP complex at endosomes, and the HOPS complex at the vacuole (Grosshans et al., 2006; Whyte and Munro, 2002). Interestingly, some tethers have been identified as Rab GEFs, including the TRAPP complex and the Vam6 subunit of the HOPS complex (Wang et al., 2000; Wurmser et al., 2000). A number of tethering complexes, which bind to specific Rab GTPases, have been characterized to date. However, since many of them harbor numerous subunits and multiple domains, their precise function remains widely elusive. We are interested in tethering within the endolysosomal system. In yeast (and later in mammalian cells), the multi- subunit HOPS/class C Vps complex was identified as a tethering complex, which is required for homotypic fu- sion at the vacuole. It consists of six proteins: Vps41 (Vam2) and Vam6 (Vps39) and the class C subunits Vps11, Vps16, Vps18, and Vps33 (Nakamura et al., 1997; Price et al., 2000b; Rieder and Emr, 1997; Seals et al., 2000; Wurmser et al., 2000). The complex interacts with the GTP form of Ypt7 (yeast Rab7) and can bind to SNAREs (Collins et al., 2005; Laage and Ungermann, 2001; Stroupe et al., 2006). It is therefore thought that HOPS mediates the transition from tethering to trans- SNARE pairing during fusion. The individual subunits have strikingly different domains. Vps33 is homologous to Sec1/Munc18 proteins, Vps11 and Vps18 have essen- tial RING domains at their C termini (Rieder and Emr, 1997), and Vps16 has two conserved domains of unknown function (Richardson et al., 2004)(http://www.pfam.org). Developmental Cell 12, 739–750, May 2007 ª2007 Elsevier Inc. 739
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Developmental Cell
Article
The CORVET Tethering Complex Interactswith the Yeast Rab5 Homolog Vps21and Is Involved in Endo-Lysosomal BiogenesisKarolina Peplowska,1 Daniel F. Markgraf,1,3 Clemens W. Ostrowicz,1,3 Gert Bange,2 and Christian Ungermann1,*1 University of Osnabruck, Department of Biology, Biochemistry Section, Barbarastrasse 13, 49076 Osnabruck, Germany2 Biochemie Zentrum der Universitat Heidelberg (BZH), Im Neuenheimer Feld 328, 69120 Heidelberg, Germany3 These authors contributed equally to this work.*Correspondence: [email protected]
DOI 10.1016/j.devcel.2007.03.006
SUMMARY
The dynamic equilibrium between vesicle fis-sion and fusion at Golgi, endosome, and vacu-ole/lysosome is critical for the maintenance oforganelle identity. It depends, among others,on Rab GTPases and tethering factors, whosefunction and regulation are still unclear. Wenow show that transport among Golgi, endo-some, and vacuole is controlled by two homol-ogous tethering complexes, the previouslyidentified HOPS complex at the vacuole anda novel endosomal tethering (CORVET) com-plex, which interacts with the Rab GTPaseVps21. Both complexes share the four classC Vps proteins: Vps11, Vps16, Vps18, andVps33. The HOPS complex, in addition, con-tains Vps41/Vam2 and Vam6, whereas theCORVET complex has the Vps41 homologVps8 and the (h)Vam6 homolog Vps3. Strikingly,the CORVET and HOPS complexe can intercon-vert; we identify two additional intermediatecomplexes, both consisting of the class Ccore bound to Vam6-Vps8 or Vps3-Vps41. Ourdata suggest that modular assembled tetheringcomplexes define organelle biogenesis in theendocytic pathway.
INTRODUCTION
Vesicle-mediated protein transport between organelles of
the endomembrane system depends on a dynamic equi-
librium between fission and fusion. Alterations of this bal-
ance lead to a loss of organelle identity and subsequently
to disease (Di Pietro and Dell’Angelica, 2005; Munro,
tion of AP-3 transport intermediates requires Vps41 function. Nat. Cell
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R.C. (2004). Mammalian late vacuole protein sorting orthologues par-
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Developmental Cell
Endolysosomal Biogenesis Linked to the CORVET Complex
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(1998). TRAPP, a highly conserved novel complex on the cis-Golgi
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cation of active HOPS complex reveals its affinities for phosphoinosi-
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Inc.
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Developmental Cell, Vol. 12
Supplemental Data
The CORVET Tethering Complex Interacts
with the Yeast Rab5 Homolog Vps21
and Is Involved in EndoLysosomal Biogenesis Karolina Peplowska, Daniel F. Markgraf, Clemens W. Ostrowicz, Gert Bange, and Christian Ungermann
Supplemental Experimental Procedures
Yeast strains and plasmids
To generate plasmids carrying GFPtagged proteins, a BamH1BglII fragment from pBSeGFP
(provided by E. Hurt, Heidelberg, Germany) was inserted into a BamH1 site of the indicated plasmid.
Plasmids were either genomically integrated by cutting with Bsp119I (pRS406NOP1prGFPVam3),
or maintained within cells by growth on selective medium (pRS415NOP1prGFPVPS41, pRS416
NOP1prGFPYCK3, pRS426NOP1prVAC8GFP). Cterminal tagging of VPS3 and VPS8 in the
indicated strains was done by integrating a PCRamplified region coding for the TAPtag (pYM13) or
the GFPtag (pYM12) and the kanamycin marker (kindly provided by M. Knop, Heidelberg, Germany)
via homologous recombination. TAPtagging of VPS41 or VPS3 was done similarly, using pBS1539 as
a template (Puig et al., 1998). VPS3, VPS8, VPS9, or the Rab GTPases VPS21, YPT7 and YPT1 were
placed under the control of the GAL1promoter using PCR fragments containing flanking regions of
the respective genes amplified from pFA6aHIS3MX6GAL1pr (VPS3), pFA6aHIS3MX6GAL1pr
3xHA (VPS8), pBS1761TRPGAL1prTAP (VPS3, VPS9), or pFA6kanMX6GAL1prGST (VPS21,
YPT7, YPT1) (Longtine et al., 1998). PHO8 and VPS21 were genomically tagged at the Nterminus
using a URA3PHO5prGFPMyc cassette, amplified from plasmid pGL (a gift from S. Munro, MRC,
Cambridge, UK; Levine and Munro, 2001). VPS21, YPT7, and YPT1 were cloned into pGEX4T3 or
pGEX2T (GE Healthscience) and purified according to the manufacturer.
2
Yeast cell lysis
After overnight growth in rich medium containing 2% glucose (YPD) or 2% galactose (YPG), cell
cultures were diluted to OD600=0.5 and incubated for 2 hours in 30°C. Cells (30 OD600 units) were
collected, washed once with DTT buffer (10 mM DTT, 0.1 M Tris/HCl pH 9.4), resuspended in 1 ml of
DTT buffer and incubated for 10 minutes in 30°C. They were then centrifuged (2 min at 4620g),
resuspended in 300 µl of spheroplasting buffer (0.16x YPD, 50 µM KPi buffer, pH 7.4, 0.6 M sorbitol),
and incubated for 20 min at 30°C in the presence of lyticase. Cells were centrifuged for 3 min at 1530g,
the pellet was resuspended in 1ml of lysis buffer (0.2 M sorbitol, 150 mM KCl, 20 mM HEPES/KOH,
pH 6.8, 1 mM DTT, 1 mM PMSF, 1xPIC) supplemented with 6 µl of 0.4 mg/ml DEAEdextran, and
incubated for 5 minutes on ice. Samples were briefly heatshocked (2 min/30°C), and unlysed cells
were removed by centrifugation at 300g for 3 min. The cell lysate was used in further experiments
(pulldowns, subcellular fractionations).
GST pull down
Cells were grown overnight in the presence of galactose to overproduce GSTRab protein
(Vps21, Ypt7 or Ypt1) together with TAPtagged Vps3 and Vps9. 200 ODs of cells were collected,
washed once with 1 ml of buffer A (20 mM Tris/HCl, pH 7.4, 150 mM NaCl, 5 mM MgCl2) and lysed
with glass beads in a presence of 300 µl of buffer A containing1xPIC and 1 mM PMSF. Lysis was
repeated twice and each time 300 µl of lysate were collected. The lysate (25 mg) was supplemented
with 0.5% Triton X100, centrifuged (30 min, 100,000g, 4°C), and then loaded onto 50 µl equilibrated
glutathione (GSH) beads. An aliquot of the lysate (0.1%) was removed as a loading control. Beads
were incubated at 4°C for 1.5 hour and then washed extensively (2 x 15 min with buffer A + 0.1%
Triton X100 and 2 x 15 min with buffer A + 0.025% Triton X100). Proteins were eluted by
3
incubating the beads for 20 min at room temperature in 600 µl elution buffer (20 mM Tris/HCl, pH 7.4,
1.5 M NaCl, 20 mM EDTA), TCAprecipitated, and analyzed by using 7.5% SDSPAGE gels and
Western blotting.
References:
Longtine, M. S., McKenzie, A., 3rd, Demarini, D. J., Shah, N. G., Wach, A., Brachat, A., Philippsen, P., and Pringle, J. R. (1998). Additional modules for versatile and economical PCRbased gene deletion and modification in Saccharomyces cerevisiae. Yeast 14, 953961.
Puig, O., Rutz, B., Luukkonen, B. G., KandelsLewis, S., BragadoNilsson, E., and Seraphin, B. (1998). New constructs and strategies for efficient PCRbased gene manipulations in yeast. Yeast 14, 11391146.
4
Figures
Figure S1. Characterization of vps3∆ mutants.
(A) Osmotic stress response. Logarithmically grown wt and vps3∆ cells were stained with FM464,
reisolated, incubated in YPD medium containing 0.4 M NaCl, and analyzed by fluorescence
microscopy after 10 and 60 minutes. Control cells did not receive salt. Quantification of at least 200
cells per condition is shown.
(B) Vacuole morphology of vps3∆ cells. BY4741 cells (wt,vps3∆ or vps8∆) were grown
logarithmically in YPD medium. To visualize vacuoles, cells were incubated for 30 minutes (pulse)
with 25 µM CMAC dye (7amino4chloromethylcoumarin) , washed, and incubated for another 30
minutes (chase) in fresh YPD before being analyzed by fluorescence microscopy.
Figure S2. Characterization of the Vps3 protein.
Localization of the Vps3 protein. BY4741 cells expressing Vps3GFP were analyzed by fluorescence
microscopy.
Figure S3. Alignment of Vps3 and Vam6 proteins from yeast and human.
The alignment was done with Jalview (Clamp, M., Cuff, J., Searle, S. M. and Barton, G. J. (2004),
"The Jalview Java Alignment Editor", Bioinformatics, 12, 4267.).
5
Figure S4: Cooverexpression of Vps3 and Vps21.
Cells cooverexpressing the indicated RabGTPase in the wt or GDPlocked form (S21N) and TAP
tagged Vps3 (3) or Vps9 (9) were grown overnight and processed for the GSH pulldown as described
in Experimental Procedures. Specifically bound proteins were eluted and analyzed by SDSPAGE and
Western blotting. The lower band in lane 3 is most likely a degradation product of Vps3 that seems to
bind efficiently to Vps21S21N. Note that Vps3 was overproduced more strongly in the absence of any
GTPase (lane 6), similarly Vps9 showed stronger expression (lane 10). A lower exposure of the load is
shown in the bottom right panel.
(C) RabGTPase present on the remaining GSH beads. Beads were boiled in SDSsample buffer and
analyzed as above. Note that Vps21S21N is poorly recovered on GSH beads, potentially due to its
decreased stability caused by the mutation.
(D) Expression of the fusion proteins in vivo. Whole cell extracts were prepared from galactose
induced cultures as described in Experimental Procedures, proteins were resolved by SDSPAGE and
analyzed by Western blotting using antiGST and ProteinAperoxidase coupled antibodies.
Figure S5. Accumulation of GFPVps21 upon Vps8 overexpression.
The experiment was done as in Figure 3F, and shows representative fields. Size bar is 10 µm.
Figure S6. Vps3 interaction with the Class C complex.
Purification of Vps3TAP from vps11∆ cells was done as described above. Eluted proteins were
analyzed by SDSPAGE and Western blotting as described before.
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Table S1. Strains used in this study
Strain Genotype Reference CUY1 BJ3505;MATa pep4::HIS3 prb1∆1.6R HIS3 lys2208 trp1∆101 ura352 gal2 can Haas et al., 1994 CUY476 BY4741;MATa his3∆1 leu2∆0 met15∆0 ura3∆0 Euroscarf library CUY473 BY4741;MATa his3∆ leu2∆ met15∆ ura3∆ vps8∆::kanMX Euroscarf library CUY765 BY4741;MATa his3∆1 leu2∆0 met15∆0 ura3∆0 vam3∆::kanMX This study CUY865 CUY476; pRS426NOP1prVAC8GFP Subramanian et al., 2006 CUY887 BY4741;MATa pRS415NOP1prGFPVPS41 This study CUY959 CUY476; PHO8::HIS5PHO5prGFPMYC LaGrassa and Ungermann, 2005 CUY1014 BY4741;MATa his3∆ leu2∆ met15∆ ura3∆ vps3∆::kanMX Euroscarf library CUY1616 BJ3505; VPS3::TAPkanMX This study CUY1792 BY4741;MATa his3∆1 leu2∆0 met15∆0 ura3∆0 VPS8::TAPkanMX This study CUY1794 CUY476; VPS3::GFPkanMX This study CUY1795 CUY476; VPS3::TAPURA3 This study CUY1796 BY4741;MATa his3∆1 leu2∆0 met15∆0 ura3∆0 vps41∆::kanMX VPS3::TAPURA3 This study CUY1797 CUY473; VPS3::TAPURA3 This study CUY1798 BY4741;MATa his3∆1 leu2∆0 met15∆0 ura3∆0 vam6∆::kanMX VPS3::TAPURA3 This study CUY1799 BY4741;MATa his3∆1 leu2∆0 met15∆0 ura3∆0 vps33∆::kanMX VPS3::TAPURA3 This study CUY1800 BY4741;MATa his3∆1 leu2∆0 met15∆0 ura3∆0 VPS41::TAPURA3 This study CUY1801 BY4741;MATa his3∆1 leu2∆0 met15∆0 ura3∆0 vam6∆::kanMX VPS41::TAPURA3 This study CUY1802 CUY1014; VPS41::TAPURA3 This study CUY1803 CUY473; VPS41::TAPURA3 This study CUY1804 BY4741;MATa his3∆1 leu2∆0 met15∆0 ura3∆0 vps33∆::kanMX VPS3::TAPURA3 This study CUY1805 BY4741;MATa his3∆1 leu2∆0 met15∆0 ura3∆0 ypt7∆::kanMX VPS3::TAPURA3 This study CUY1806 BY4741;MATa his3∆1 leu2∆0 met15∆0 ura3∆0 vps21∆::kanMX VPS3::TAPURA3 This study CUY1819 CUY473; VPS21::URA3PHO5prGFP This study CUY1820 BY4741;MATa his3∆ leu2∆ met15∆ ura3∆ vam6∆::kanMX VPS21::GFPURA3 This study CUY1826 CUY1014; VPS21:: URA3PHO5pr GFPMYC This study CUY1836 CUY1014; PHO8::URA3PHO5prGFPMYC This study CUY1837 CUY1014; pRS426NOP1prGFPVAC8 This study CUY1838 CUY476; URA3::pRS406NOP1prGFPVAM3 This study CUY1839 CUY1014; URA3::pRS406NOP1prGFPVAM3 This study CUY1841 CUY476; pRS416NOP1prGFPYCK3 (URA3) This study CUY1842 CUY1014; pRS416NOP1prGFPYCK3 (URA3) This study CUY1845 CUY1794; vps8∆::URA3 This study CUY1847 CUY1800; VPS3::HIS3GAL1pr This study CUY1849 CUY1014; pRS415NOP1prGFPVPS41 (LEU2) This study CUY1850 BY4741;MATa his3∆ leu2∆ met15∆ ura3∆ vps8∆::kanMX VAM3::URA3 This study CUY1877 BY4741;MATa his3∆ leu2∆ met15∆ ura3∆ VPS8::TAPkanMX vps33∆::URA3 This study CUY1878 BY4741;MATa his3∆ leu2∆ met15∆ ura3∆ VPS3::TAPURA3 vps11∆::kanMX This study CUY1883 BY4741;MATa his3∆ leu2∆ met15∆ ura3∆ VPS41::TAPkanMX VPS8::HIS3GAL1pr3HA This study CUY1895 BY4733;MATalpha; his3∆200 leu2∆0 met15∆0 trp1∆63 ura3∆0 VPS3::TRP1GAL1prTAP This study CUY1897 BY4741;MATa his3∆ leu2∆ met15∆ ura3∆ vam6∆::kanMX VPS8::HIS3GAL1pr3HA
VPS41::TAPURA3 This study
CUY1915 BY4741;MATa his3∆1 leu2∆0 met15∆0 ura3∆0 VPS3::kanMXGAL1pr This study CUY1936 CUY1895; VPS21::kanMXGAL1prGST This study CUY1949 CUY1895; VPS21 S21N::kanMXGAL1prGST This study CUY1948 CUY1895; YPT1::kanMXGAL1prGST This study CUY1952 BY4719;MATa trp1∆63 ura3∆0 VPSs8::kanMX VPS21::URAPHO5prGFP pV2RFPYPT7 (TRP1) This study CUY1960 BY4719;MATa trp1∆63 ura3∆0 vps8∆::kanMX VPS21::URAPHO5prGFP pV2RFPYTP7(TRP1) This study CUY1967 BY4719;MATa trp1∆63 ura3∆0 VPS21::URA3PHO5prGFP This study CUY1969 BY4733;MATalpha; his3∆200 leu2∆0 met15∆0 trp1∆63 ura3∆0 VPS9::TRP1GAL1TAP
VPS21::kanMXGAL1prGST This study
CUY1972 BY4719;MATa trp1∆63 ura3∆0 VPS21::URA3PHO5prGFP pV2RFPYPT7 (TRP1) This study CUY2251 BY4733;MATalpha his3∆200 leu2∆0 met15∆0 trp1∆63 ura3∆0 VAM6::kanMXGAL1pr
VPS21:: URA3PHO5prGFP This study
CUY2252 CUY1915; VPS21::URA3 PHO5prGFP This study CUY2253 BY4733;MATalpha his3∆200 leu2∆0 met15∆0 trp1∆63 ura3∆0 VPS8::HIS3GAL1pr3HA
VPS21:: URA3PHO5prGFP This study
CUY2359 BY4719;MATa trp1∆63 ura3∆0 vps8∆::kanMX VPS3::TRP1GAL1prTAP This study CUY2278 CUY1826; VPS8::HIS3GAL1pr3HA This study