Welcome to the Reinisch Laboratory
Research: Structural Studies in Membrane Trafficking
Ours is a structural biology group whose overall goal is to better understand the mechanisms underlying membrane trafficking processes—i.e., how vesicles transport material between different membrane-bound organelles within the cell or to and from the plasma membrane.
Our most recent research has centered primarily on the fundamental question of how membrane identity is achieved, thus assuring cargo delivery to the correct destination. The lipid composition of a membrane, and in particular the enrichment of specific phosphoinositide lipids at a membrane, is a major determinant of its identity, enabling the recruitment of proteins that recognize specific lipid headgroups (1,2). During the last 3-4 years, we have begun to investigate how the composition of lipid bilayers is regulated, in particular as pertaining to the levels of the phosphoinositides. That is, how are the activities of phosphoinositide kinases and phosphatases at different organelles are regulated?
A major effort in the laboratory is directed toward understanding phosphatidylinositol 4-phosphate (PI4P) metabolism, and one of our main projects, which is a collaboration with the group of my colleague Pietro De Camilli, concerns the Stt4/PI4KIII lipid kinase complex. This complex is conserved in eukaryotes and synthesizes PI4P at the plasma membrane. Our structural studies so far have formed the basis for understanding how the kinase is recruited to the plasma membrane by the scaffolding proteins Efr3 and Ypp1/TTC7 and led to initial insights as to how complex assembly may be regulated to modulate kinase activity there (3). Our current effort is directed at understanding how Ypp1/TTC7 stimulates kinase activity. In collaboration with my colleague C.G. Burd, we have also obtained insights as to how PI4P levels at the Golgi apparatus are regulated, obtaining a crystal structure of a complex comprising Vps74/GOLPH3 and Sac1. Sac1 is a PI4P phosphatase which resides primarily at the ER, whereas Vps74/GOLPH3 localizes to the Golgi. The structure explains how Sac1 can be recruited to the Golgi by Vps74/GOLPH3 (4) to regulate PI4P levels there.
It is becoming increasingly clear that membrane lipid composition is also modulated at membrane contact sites, where two organelles are apposed closely enough so that non-vesicular lipid exchange between their membranes is possible (5,6). The molecular mechanisms underlying lipid exchange, however, remain unexplored. The Extended-Synaptotagmin proteins were previously characterized as tethers that are localized to and maintain ER-PM contact sites (7,8). Our crystal structure of Extended-Synaptotagmin2 (E-Syt2) demonstrated that a protein module of previously unknown function, the SMP domain, within E-Syt2 is a lipid binding module, strongly supporting that lipid exchange at these contact sites is mediated by lipid transfer proteins (9). Because SMP-domains are also found at other contact sites (for example, in the ERMES complex at ER-mitochondrial sites) our findings have broad implications for these sites also. This work was a collaboration with my colleague P. De Camilli.
Mechanisms for activating Rab GTPases.
Besides the phosphoinosities, a second major determinant of membrane identity is the subset of activated Rab GTPases present at a particular membrane (1,2). To better understand how Rabs are activated, we have obtained crystal structures for complexes of Rabs bound to their activating guanine nucleotide exchange factor (GEF). These include Sec2/Sec4 (10), TRAPPI/Ypt1 (11,12), and most recently, a DENND1B/Rab35 complex, where DENN-domain proteins are the largest GEF family identified to date (13). Interestingly, although these GEFs all have different folds, the mechanisms underlying GTPase activation are similar. In most cases, GEF binding causes restructuring of the Rab switch regions. This removes an aromatic residue that normally stabilizes bound nucleotide from the nucleotide binding pocket, lowering the affinity of the Rab for nucleotide; and it opens the nucleotide binding pocket to solvent, facilitating nucleotide exchange. Our work on Rab activation involved collaborations with F. Barr (Oxford), S. Ferro-Novick (UCSD), or P. Novick (UCSD).
Regulation of Membrane Fusion.
Previously, we contributed to understanding the architecture of multi-component tethering complexes in the CATCHR family, which are thought to promote the assembly of the SNARE complex and other membrane fusion machinery. We discovered that CATCHR complex subunits have similar folds in that they are all composed of -helical bundles assembled into rods (14) and demonstrated that the GARP complex is a member of the CATCHR family (15). We also studied regulation of SNARE assembly at the neuronal synapse, in particular how the protein complexin interacts with assembling SNARE complexes there so as to regulate secretory vesicle fusion (16,17). Our crystal structure of complexin with an incompletely assembled SNARE complex led to a model for how this protein is able to inhibit SNARE assembly pending an action potential. We collaborated with P. Novick (UCSD) on work regarding CATCHR complexes and my colleague James Rothman for work regarding complexin.
(1) Di Paolo, G., De Camilli, P. Phosphoinositides in cell regulation and membrane dynamics. Nature 2006, 443:651-657.
(2) Behnia, R., Munro, S. Organelle identity and the signposts for membrane traffic. Nature 2005, 438:597-604.
(3) Wu, X., Chi, R.J., Baskin, J.M., Lucast, L., Burd, C.G., De Camilli, P., Reinisch, K.M. Structural insights into assembly and regulation of the plasma membrane PI 4-kinase complex. Dev. Cell 2014, 28:19-29
(4) *Cai, Y., *Deng, Y., Horenkamp, F., *Reinisch, K.M., *Burd, C.G. Sac1-Vps74 structure reveals a mechanism to terminate phosphoinositide signaling in the Golgi apparatus. JCB 2014, 206:485-491. * Co-first or co-corresponding authors.
(5) Helle, S.C., Kanfer, G., Kolar, K., Lang, A., Michel, A.H., Kornmann, B. Organization and function of membrane contact sites. Biochim Biophys Acta 2013, 1833: 2526-2541.
(6) Munro, S. Open questions: what is there left for cell biologists to do? BMC Biol 2013, doi:10.11.1186/1741-7007-11-16.
(7) Manford, A.G., Stefan, C.J., Yuan, H.L., Macgurn, J.A., Emr, S.D. ER-to-plasma membrane tethering proteins regulate cell signaling and ER morphology. Dev Cell 2012, 23: 1129-1140.
(8) Giordano, F., Saheki, Y., Idevall-Hagren, O., Colombo, S.F., Pirruccello, M., Milosevic, I., Gracheva, E., Bagriantsev, S.N., Borgese,N., De Camilli, P. PI(4,5)P2-dependent and Ca2+-regulated ER-PM interactions mediated by the extended synaptotagmins. Cell 2013, 153: 1494-1509.
(9) Schauder, C.M., Wu, X., Saheki, Y., Narayanaswamy, P., Torta, F., Wenk, M.R., *De Camilli, P., *Reinisch, K.M. Structure of a lipid-bound extended synaptotagmin indicates a role in lipid transfer. Nature 2014, 10:552-555. *co-corresponding authors.
(10) Dong, G., Medkova, M., Novick*, P. & Reinisch*, K.M. A catalytic coiled-coil: activation of the Rab GTPase Sec4p by Sec2p. Mol. Cell 2007, 25:455-462. *co-corresponding authors. Highlighted in F1000.
(11) Cai, Y., Chin, H.F., Lazarova, D., Menon, S., Fu, C., Sclafani, A., Cai, H., Rodgers, D.W., De La Cruz, E.M., Ferro-Novick*, S. & Reinisch*, K.M. The structural basis for the activation of the Rab Ypt1p by the TRAPP membrane tethering complexes. Cell 2008, 33:1202-1213. *co-corresponding authors. Previewed in Cell.
(12)*Tan, D., *Cai, Y., Wang, J., Zhang, J., Menon, S., Chou, H.T., *Ferro-Novick, S., *Reinisch, K.M., *Walz, T., The EM structure of the TRAPPIII complex leads to the identification of a requirement for COPII vesicles on the macroautophagy pathway. PNAS 2013, 110:19432-19437. *Co-first or co-corresponding authors.
(13) Wu, X., Bradley, M.J., Kümmel, D., Cai, Y., De La Cruz, E.M., Barr, F.A., Reinisch, K.M. Insights regarding guanine nucleotide exchange from the structure of a DENN-domain protein complexed with its Rab GTPase substrate. PNAS 2011, 108:18672-18677.
(14) Dong, G., Hutagalung, A.H., Fu, C., Novick*, P. & Reinisch*, K.M. The structures of exocyst subunit Exo70p and the Exo84p C-terminal domains reveal a common motif. Nat. Struct. Mol. Biol. 2005, 12:1094-1100. *co-corresponding authors.
(15) Vasan, N., Hutagalung, A., Novick, P., and Reinisch, K.M. Structure of a C-terminal fragment of its Vps53 subunit suggests similarity of Golgi-associated retrograde protein (GARP) complex to a family of tethering complexes. PNAS 2010, 107:14176-14181.
(16) Kümmel, D., Krishnakumar, S.S., Radoff, D.T., Li, F., Giraudo, C.G., Pincet, F., *Rothman, J.E., *Reinisch, K.M. Complexin cross-links pre-fusion SNAREs into a zig-zag assembly. NSMB 2011, 18:927-933. *co-corresponding authors.
(17) Krishnakumar, S.S., Radoff, D.T., Kümmel, D., Giraudo, C.G., Li, F., Khandan, L., Baguley, S.W., Coleman, J., Reinisch, K.M., Pincet, F., Rothman, J.E. A conformational switch in complexin is required for synaptotagmin to trigger membrane synaptic fusion. NSMB, 2011, 18:934-940.