Regulation of Membrane Trafficking, Cytoskeleton Dynamics, and Cell Polarity by ROP/RAC GTPases

Rho of plants (ROP) proteins, also known as RAC proteins, are Rho-related GTPases that function as molecular switches in a multitude of signaling cascades involved in the regulation of the actin and microtubule cytoskeleton, of vesicle trafficking, and of plant responses to hormones, stresses, or light (Yang, 2002; Berken, 2006; Nibau et al., 2006; Yang and Fu, 2007). Rho GTPases are Ras-related small guanine nucleotide-binding proteins (G-proteins) that bind GTP and GDP with high affinity and hydrolyze GTP inefficiently. Rho GTPases switch between GTP-on to GDP-off states by highly regulated GDP/GTP exchange and GTP hydrolysis (Bourne et al., 1991; Vetter and Wittinghofer, 2001). Only in the GTP-bound state can Rho GTPases interact with effectors to elicit downstream signaling. The GDP/GTP exchange is catalyzed by guanyl nucleotide exchange factors (GEFs), and GTP hydrolysis is enhanced by GTPase-activating proteins (GAPs). Like other members of the Ras superfamily of small G-proteins, Rho GTPases are soluble proteins that associate with and function at cell membranes by virtue of the posttranslational lipid modifications prenylation and S-acylation (Hancock et al., 1989; Michaelson et al., 2001). A third group of regulating proteins are Rho guanyl nucleotide dissociation inhibitors (RhoGDIs), which inhibit GDP/GTP exchange and facilitate the cycling of Rho GTPases on and off membranes (DerMardirossian and Bokoch, 2005). The ability of Rho GTPases to interact with membranes allows these proteins to regulate actin polymerization and vesicle trafficking at discrete sites of the plasma membrane and of internal membranes, which is essential for their role in the control of cell polarity (Ridley, 2006). As part of the Plant Physiology focus issue on membrane biology, this review focuses on subcellular targeting of plant ROP/RAC GTPases and on the role of these proteins in the regulation of membrane trafficking, cytoskeleton organization, and cell polarity. Other aspects of ROP/RAC biology, such as the role of these GTPases in hormonal or stress signaling, will only be summarized in brief. We refer interested readers to several excellent recent reviews on ROP/RAC GTPases that highlight these other topics (Molendijk et al., 2004; Xu and Scheres, 2005; Nibau et al., 2006; Yang and Fu, 2007; Berken and Wittinghofer, 2008; Kost, 2008). Throughout this review, we have opted to use the ROP nomenclature for the sake of clarity. However, we use the RAC terminology in instances in which there is no ROP nomenclature or when publications have used the term RAC rather than ROP. Finally, we apologize to those colleagues whose work we have not been able to cite due to lack of space. Based on cell biological studies in animals, the Rho superfamily was initially divided into three major subfamilies designated Rho, Rac, and Cdc42 (Ridley and Hall, 1992; Ridley et al., 1992; Hall, 1998). With the increased availability of sequence information, the Rho family has been expanded and is currently suggested to include eight to nine subfamilies (Boureux et al., 2007; Vega and Ridley, 2007). Two methods of ROP/RAC classification are described in the literature. One classification placed ROP/RAC GTPases as a branch in the Rac subfamily and divided them into two subgroups, designated type I and type II, according to the structure of the C-terminal hypervariable domain (Winge et al., 1997). The second classification method, which is based on nucleotide sequences, suggested that ROP/RAC GTPases diverged as a separate group prior to the separation between Rac and Cdc42 and can be divided into four subgroups, which were designated I, II, III, and IV. Subgroups I and II correspond to type II ROP/RAC and and correspond to type I ROP/RAC GTPases of the classification (Yang, 2002; et al., et al., sequence of the Rho superfamily in (Boureux et al., the in the literature. that Rac GTPases were the family of Rho GTPases and that the Rho and Cdc42 diverged the Rac family in The Rho subfamilies were by of the family in in (Boureux et al., 2007). are ROP/RAC GTPases are to Rac, diverged as a separate Rac group prior to the of Rac, Rho, and The in of et al., and et al., the that ROP/RAC GTPases a of the Rho GTPases and Wittinghofer, 2008). this review, the classification of ROP/RAC GTPases into type I and type II according to their hypervariable domain will be used to in lipid and subcellular targeting between these two to other Rho ROP/RAC GTPases a which is for GTP and for with proteins, and a hypervariable which subcellular ROP/RAC GTPases have a molecular of to and of of et al., and et al., that these ROP/RAC GTPases a of by four The and are by that highly for and GTP hydrolysis (Bourne et al., 1991; Vetter and Wittinghofer, Berken and Wittinghofer, 2008). Like in Ras and or the of ROP/RAC proteins and them et al., and Kost, 2006; Berken and Wittinghofer, 2008). highly or the affinity of ROP/RAC GTPases for guanine their with et al., Berken et al., 2005; Berken and Wittinghofer, 2008). in Ras and ROP/RAC have by with A sequence of and ROP/RAC GTPases has the and that four are highly et al., As will be this review, and have been to ROP/RAC to the and hypervariable Rho proteins a domain the that is suggested to interact with effectors and that in and the domain is two and four with and Rho GTPases et al., 2006; et al., 2007). As in members of the Rho the hypervariable domain is at the C-terminal of ROP/RAC type I ROP/RAC the hypervariable domain of a which is by I et al., and a The hypervariable domain of type II ROP/RAC GTPases has a structure and membrane by a that S-acylation et al., et al., 2002; and 2006). ROP/RAC subcellular has been by and cell have that these GTPases are with the plasma membrane in a of cell and enhanced membrane at sites in and et al., et al., et al., 2002; et al., 2002; et al., 2007; et al., 2005; and 2006; et al., 2007). ROP/RAC GTPases were in the membrane of of et al., were in the membrane and the of et al., lipid modifications of type I and type II ROP/RAC is at the plasma membrane of by membrane on a and by ROP/RAC GTPases are the soluble the membrane these proteins between and and S-acylation the of and prenylation or S-acylation is for the membrane of type I or type II ROP/RAC The and are at the of ROP/RAC and the lipid of these is the three C-terminal of the of type I ROP/RAC GTPases are and the group of the is into the membrane or bind to proteins et al., 2006; et al., which to be in to the lipid type I and type II GTPases a which to membrane to I ROP/RAC GTPases are in the are to the for and type I ROP/RAC GTPases be the to the plasma membrane the or by a of at type I ROP/RAC GTPases is which in of these proteins into and membrane known as type II ROP/RAC GTPases are to the plasma membrane by virtue of S-acylation of two or in the C-terminal S-acylation of this domain not on the ROP/RAC and at the plasma The of type I ROP/RAC GTPases is in the by et al., et al., 2007). proteins two posttranslational to as The of these modifications of the three by of two and the group of the is by and et al., of have been and in et al., et al., 2002; et al., 2002; et al., to their and the and are at the et al., et al., that prenylation in the type I ROP/RAC GTPases are to the is the to the plasma membrane the or by a type I GTPases between membrane and et al., 2007). was in and GDP/GTP of ROP/RAC proteins between and et al., 2007). by that was only or was and by and et al., 2007). S-acylation at highly the et al., and in the GTP-bound the are in and Rho S-acylation of these in proteins has not been to The hypervariable domain of type II ROP/RAC GTPases of a sequence designated the and a The is of two that S-acylation et al., 2002; and and that are by to The are by to the S-acylation of the described S-acylation is et al., 2002; and 2006). with the the between the and the domain are for membrane of type II ROP/RAC GTPases and 2006). The domain has two essential prenylation by of et al., et al., and ROP/RAC membrane et al., 2002; and 2006). is that in proteins function as with which has not been in plant to and with et al., 2005; et al., 2006; and 2006; et al., 2007; et al., 2008). has been that the of Rho proteins and other small GTPases interact with and et al., 2006). A in with in a a highly that membrane of a et al., 2005). the Cdc42 with in the membrane a domain and with Cdc42 a The with is for of and for function in cell et al., 2008). A for ROP/RAC function cell GTP-bound ROP/RAC GTPases at the plasma membrane at the of based on lipid of the hypervariable of this domain with the membrane lipid ROP/RAC with the plasma membrane at the of the with at the to the of highly ROP/RAC and membrane trafficking for cell the of signaling the of in of which is by with the plasma membrane at the of the and in the control of ROP/RAC organization, and membrane the of which to the of which to the of by and a of signaling summarized in The of ROP/RAC on the cycling of ROP/RAC GTPases between and which is based on the GTP hydrolysis by these proteins, the separation of and sites of to the of and into discrete membrane prenylation the between Rho proteins and et al., and Bokoch, 2005). the of and was to into a by the of the et al., et al., et al., 2001). their plant are to function to their in other and Wittinghofer, 2008). II ROP/RAC GTPases are not in plants et al., that be regulated by a in type II ROP/RAC GTPases are than the type I proteins et al., S-acylation of in the hypervariable domain with et al., 2001). be that S-acylation of type I ROP/RAC GTPases their for with Rho with to factors such as et al., and proteins, et al., The on the S-acylation of and in have and in to which is based on to S-acylation was suggested to role in signaling and to as lipid are and membrane that of proteins their lipid were suggested to function as signaling that can their and in to and of in plants their in and in are in and proteins a type I ROP/RAC et al., 2004; et al., 2005; et al., 2006). proteins into et al., S-acylation of and other ROP/RAC GTPases to can interact with other GTP-bound which was to be and in et al., is that type I ROP/RAC GTPases these membrane The lipid is is that are of biological membranes 2006; 2007). A for has been that to the lipid 2006). The that lipid are that are by their proteins and which in proteins to to this lipid are that and with this in the and of and are and that in the S-acylation of ROP/RAC GTPases be for proteins and to discrete membrane based on of the in that of Ras a that into The that Ras in to the of the a signaling the The of the which is in is into a by the Ras The is Ras to the The is to be on Ras et al., 2007). a function in ROP/RAC signaling in be the of ROP/RAC GTPases in at the of that was and et al., 2005; et al., 2006). has been to ROP/RAC GTPases et al., be of to into is involved in the of ROP/RAC signaling by this of Rho, Rac, and Cdc42 in that cycling between and with to the of in with are essential for rather than et al., 2006; et al., 2007). of Cdc42 function in that the GTP with of Cdc42 to the are for and for the of a and 2008). that discrete of cycling of Rho proteins between and states and of the is not to cell polarity. the Rho switch that the and of with the separation of the and the for cell with the studies have that the of of type I and type II ROP/RAC GTPases cell et al., et al., et al., 2002; et al., et al., 2002; et al., et al., 2005). the factors involved in ROP/RAC and downstream signaling, which is based on studies in and ROP/RAC proteins at the plasma membrane of the et al., et al., et al., ROP/RAC was also of et al., 2005). was to at the of the and Kost, 2006; in the ROP/RAC the plasma membrane to the and a of in was to the of the to et al., 2006). that the of the to the is for ROP/RAC and at the of to be involved in the of ROP/RAC and that ROP/RAC at a domain of the plasma which is by in the (Molendijk et al., et al., 2005; et al., 2006). ROP GTPases regulate (Molendijk et al., et al., 2002; et al., 2005). in the as a of enhanced and ROP/RAC at the plasma membrane et al., 2005). that is essential for the of ROP/RAC and cell also with the function in the control of ROP/RAC in a than of the ROP/RAC proteins in were in the the were in the soluble et al., in and were not in soluble separation and membrane et al., 2007). ROP/RAC targeting to on function in the soluble ROP/RAC in other cell to be the of in these cell of small of soluble ROP/RAC GTPases cycling between the and the plasma membrane such as which can between membranes and membranes, or internal which of in of the plasma a family of ROP nucleotide their domain et al., 2005; et al., 2006). of between the domain of the and the family the on of ROP/RAC GTPases be et al., 2005; and 2006). to with ROP/RAC GTPases to the plasma membrane at the et al., which function of these proteins in ROP/RAC their by and 2007). However, to be function as that and ROP/RAC GTPases to membrane the described and the be suggested ROP/RAC GTPases are to highly membrane for by a as in the of et al., 2006). of in the of proteins that and and ROP/RAC ROP/RAC is and regulated by the and The of ROP/RAC is a et al., et al., and in the of a and Kost, 2006). in separation of to the of the to the of ROP/RAC GTPases to the can ROP/RAC GTPases the membrane and function to facilitate highly ROP/RAC et al., 2005; et al., 2006). the regulation of the actin and microtubule cytoskeleton, and of membrane trafficking, ROP/RAC GTPases the of polarity. function of Rho GTPases in and is the control of membrane Rho GTPases at the plasma membrane at sites of cell for this and 2007). Rho GTPases are not only with the plasma membrane also with the and these Rho GTPases or regulate for and or to the plasma membrane and Ridley, 2006). the molecular by which Rho GTPases control membrane trafficking in and are not a role in these and Rho GTPases with or with proteins that the et al., signaling the of actin proteins such as et al., 2005). of or Rho GTPases is to in the of these can the of at membranes, or inhibit vesicle with membranes, or facilitate the of or membrane the et al., 2004; Ridley, 2006). and Rho GTPases not only also by with and of the The is in the of which the of these with the plasma membrane et al., and 2007). and Cdc42 interact with and of the et al., and 2002; et al., 2005). are to be for the targeting of the and for lack the domain in et al., the Rho with in and this to lipid in the plasma in of the to the plasma membrane in to et al., by which Rho GTPases membrane trafficking include the or with proteins involved in the and of et al., et al., and the of lipid for of the signaling lipid et al., is a of membrane trafficking, which to have in the of and as as in the of with the plasma membrane et al., and and to the targeting of the in et al., 2007; et al., 2008). also Rho membrane and to and the of a of actin proteins et al., or be by to which to into the the of with the plasma membrane in et al., The of plant has been to on internal by in the and on of cell can cell in other than the recent has that and membrane trafficking by ROP/RAC GTPases also in the of plant and 2005; et al., 2006; 2006). plant in to or cell has been to be with a of actin and the which a and with the plasma membrane at sites et al., et al., 2005). and are highly with of to than and at the in a based on a known as actin in the that are for the and at the are essential for the of these 2008). in to include a or et al., et al., the plasma membrane et al., 2001). of these were to the of to sites of their with the plasma membrane et al., et al., the and function of in is A to the the plasma membrane in of has not been in plant that membrane by actin polymerization not a role in the of these to membrane trafficking for the of cell in the at sites plant and A of has that the of for cell to the on the of with the plasma membrane at a than is for plasma membrane role not only of also of membrane in the of plant et al., of is in of of is of is into a of a plant to was not into of plants a of at and with et summarized in the that ROP/RAC GTPases control cell by regulating membrane trafficking the control of actin and function of in membrane trafficking has also been The of the of by the was to of actin the of actin are to membrane trafficking for the of cell and of at this et al., 2005). of in plants was to the of the of membrane in and to with the of A in these and other et al., 2005). are in plant with the by the of and have been to plasma membrane proteins and that et al., 2002; et al., et al., The of ROP/RAC on by are in and in to was A and was not at in to et al., 2005). these a role of ROP/RAC GTPases in the control of membrane the function of lipid in the regulation of in and will be to the of ROP/RAC GTPases in has a function in the control of membrane in between ROP/RAC GTPases and the control of membrane trafficking was by the that of to a of the et al., 2007). is a of a family of proteins that interact with ROP/RAC GTPases et al., 2007). is to function as a to the plasma membrane by Cdc42 was to the of et al., and 2002; et al., 2008). eight have been in et al., and the of the was et al., 2008). of to et al., is in and et al., 2005; et al., and of cell are in et al., 2006). that the plant is for cell of essential function in Rho domain and not bind to ROP/RAC GTPases et al., 2007). However, was to interact with and ROP/RAC GTPases were to to the plasma membrane et al., 2007). plants and a to that of in et al., 2007). to be is in this However, that the regulation of function and membrane trafficking by ROP/RAC GTPases their with role in the control of cell in plants et al., 2007). ROP/RAC signaling to regulate and to cell One of these the with the and Other are based on ROP/RAC effectors or on proteins such as in for these factors have on cell the of these factors is in this and the are that the are of a and three with the cell for the of the of these are not are to in part on cell and and the actin cytoskeleton 2005; 2005). The of with organization, and are by the of with that with et al., et al., are in of of the or the 2005; 2005). Rho the of the to The that ROP/RAC GTPases interact with of the suggested that also in plant is by Rho signaling et al., 2004; et al., 2007). by in the the to branch et al., and that ROP/RAC cell of the and et al., 2008). was also to interact with proteins et al., 2007; et al., that the function as a that with ROP/RAC and the to the is to membrane by ROP/RAC that in the cytoskeleton is than et al., that ROP/RAC GTPases also control in these by or control of actin of has in to these plants cell and cell which are in the of such as and 2005; 2005). The in cell that and in membrane trafficking for the of cell 2005). plants of which are at plants and a cell and are in that ROP/RAC GTPases by have in the control of the of a of cell on the to regulate actin and membrane trafficking, in and other and are the and have only on et al., 2002; 2005; that ROP/RAC or signaling are not essential for a family of ROP/RAC effectors that a domain of this domain with other or with other proteins et al., 2001). are also in effectors of and Rho GTPases and to GTP-bound Rho proteins et al., 2001). have been to in the control of cell downstream of ROP/RAC and have been to the control of cell in in the of et al., 2005). and at the plasma in by these these GTPases to the of a of actin the plasma membrane in and also to bind and which can associate with and is to the of highly microtubule in between to have the ability to and Based on these and colleagues have that a between and which of actin and highly microtubule in the cell is for the control of cell by and studies et al., have that and are in the of at are will be to the of ROP/RAC proteins, and the in the control of at these Two have also been to in the control of by ROP/RAC GTPases et al., 2005). to the in by which at the plasma was suggested to the of a of actin at the of plant on a As in in the et al., are to at the of plant regulation et al., of to into the at the which of the the was to the in with the that the of cell as as are control of this on a between the and et al., 2005). at the which into the at high have also been to role in the control of with and signaling et al., 2008). and as ROP/RAC effectors in the regulation of cell the molecular to control and membrane trafficking to be Like and Rho ROP/RAC GTPases can also by regulating The with in and a the et al., in actin cell and by ROP/RAC ROP/RAC in enhanced of at which the ability of this to associate with inhibit cell and et al., to the in ROP/RAC can not only the of the also by and of which in to Rho in are not in plant was to be by et al., 2001). As Rho GTPases the or to be to and other in is to that of role in the control of the of other in these of a family of proteins which is for the of and have been to such in to in and 2006). of these is with the cell a the of a cell between et al., 2005). is essential for the of a role in the vesicle trafficking to and the cell that is for the function of this structure 2005). A of plant which to be to those of or to Rho is to of these proteins downstream of ROP/RAC in the control of actin and membrane ROP/RAC GTPases have been to bind to and et al., 2007). et al., and in et al., for the of was to be essential for cell the of plasma membrane are to be essential for the of a for et al., at the were to in with in and in cell et al., 2007). to regulating actin and membrane trafficking, at the were to a that is to to the of the polarity of et al., 2008). of a are to ROP/RAC and to is also enhanced and in these that for is the control of ROP/RAC GTPases et al., 2005). As Rho ROP/RAC GTPases interact with which the signaling lipid et al., ROP/RAC GTPases and to high at the of et al., et al., and et al., 2007; et al., 2008). by of to the domain of et al., that as a ROP/RAC in which vesicle with the plasma membrane by or ability to the of actin of of also at the plasma membrane of et al., 2007; and known to be by to regulating membrane trafficking as a ROP/RAC in ROP/RAC as part of that to ROP/RAC signaling and cell at the of 2008). hydrolysis by with the plasma membrane at the of the to be to the of this lipid to the et al., 2006; et al., 2006). the lipid of also at the on that this lipid to the the of at the of the et al., 2006). The of by is a and signaling in and of this not to in plants and The of of in the control of membrane trafficking in the of factors whose is by this hydrolysis also a soluble known for ability to into the of et al., et al., and other will be to the that to the of the which to have in the control of membrane trafficking in of the summarized ROP/RAC signaling to the control of membrane trafficking, that membrane trafficking in also ROP/RAC signaling. As other Rho proteins, type I ROP/RAC GTPases at the are to the plasma the The of by ROP/RAC with the of these proteins on the of to sites of their at the plasma a that to the of As by the of the of the to the membrane or regulate the of factors involved in ROP/RAC signaling, or the control of such by ROP/RAC signaling excellent for or regulation that a role in the control of cell with the of membrane trafficking and Rho signaling, by the of type I ROP/RAC at the plasma membrane of which is for (Molendijk et al., 2001). However, not the of in or at the plasma membrane et al., 2005). that the targeting of this and other type II ROP/RAC not on membrane of the of membrane trafficking by ROP/RAC GTPases is that the membrane with which these proteins are are the of membrane trafficking is and ROP/RAC the to the was to be to for the of ROP/RAC by the of with the plasma membrane 2008). with of the of ROP/RAC signaling and membrane trafficking in is by regulation rather than by the of polarity with have in of the regulation of ROP/RAC function and of the downstream signaling by their Rho regulation and downstream signaling have highly plants have a of involved in these ROP/RAC by signaling by and and of these in the signaling a function of Rho proteins, ROP/RAC to be the control of membrane trafficking, or signaling ROP/RAC signaling at discrete of the plasma membrane ROP/RAC GTPases regulate actin and downstream of ROP by the ROP/RAC actin and actin ROP/RAC GTPases also bind to and which this the of The to the ROP to the control of in cell by that this also to the regulation of of by ROP/RAC GTPases in which into the by which a ROP/RAC also are to and to the of with the plasma was also to the of a plant responses to et al., ROP/RAC GTPases interact with which signaling lipid has the of vesicle and the regulation of proteins, and the of of the ROP/RAC with and by to the of ROP/RAC also ROP/RAC membrane and which and which are involved in signaling of such is by the and of ROP/RAC signaling. A of and is to the and involved in ROP/RAC signaling to membrane trafficking this will be essential to in the ROP/RAC GTPases in and to the that of them be with part of this will be the of lipid modifications of ROP/RAC GTPases and of the of these modifications on as as on and ROP/RAC to be and their targeting to be will be to the of ROP/RAC the membrane trafficking involved in plant cell and the role of in these based on of the of or ROP/RAC were in the of