Functions of Vrp1p in cytokinesis and actin patches are distinct and neither requires a WH2/V domain
2001; Springer Nature; Volume: 20; Issue: 24 Linguagem: Inglês
10.1093/emboj/20.24.6979
ISSN1460-2075
Autores Tópico(s)Fungal and yeast genetics research
ResumoArticle17 December 2001free access Functions of Vrp1p in cytokinesis and actin patches are distinct and neither requires a WH2/V domain Thirumaran Thanabalu Thirumaran Thanabalu Institute of Molecular Agrobiology, 1 Research Link, The National University of Singapore, Singapore, 117604 Republic of Singapore Present address: School of Biological Sciences, Nanyang Technological University, 1 Nanyang Walk, Singapore, 637616 Republic of Singapore Search for more papers by this author Alan L. Munn Corresponding Author Alan L. Munn Institute of Molecular Agrobiology, 1 Research Link, The National University of Singapore, Singapore, 117604 Republic of Singapore Department of Biochemistry, The National University of Singapore, 10 Kent Ridge Crescent, Singapore, 119260 Republic of Singapore Search for more papers by this author Thirumaran Thanabalu Thirumaran Thanabalu Institute of Molecular Agrobiology, 1 Research Link, The National University of Singapore, Singapore, 117604 Republic of Singapore Present address: School of Biological Sciences, Nanyang Technological University, 1 Nanyang Walk, Singapore, 637616 Republic of Singapore Search for more papers by this author Alan L. Munn Corresponding Author Alan L. Munn Institute of Molecular Agrobiology, 1 Research Link, The National University of Singapore, Singapore, 117604 Republic of Singapore Department of Biochemistry, The National University of Singapore, 10 Kent Ridge Crescent, Singapore, 119260 Republic of Singapore Search for more papers by this author Author Information Thirumaran Thanabalu1,3 and Alan L. Munn 1,2 1Institute of Molecular Agrobiology, 1 Research Link, The National University of Singapore, Singapore, 117604 Republic of Singapore 2Department of Biochemistry, The National University of Singapore, 10 Kent Ridge Crescent, Singapore, 119260 Republic of Singapore 3Present address: School of Biological Sciences, Nanyang Technological University, 1 Nanyang Walk, Singapore, 637616 Republic of Singapore *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:6979-6989https://doi.org/10.1093/emboj/20.24.6979 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Vrp1 (verprolin, End5) is a Saccharomyces cerevisiae actin-associated protein and is related to mammalian Wiskott–Aldrich syndrome protein (WASP)-interacting protein (WIP). Vrp1-deficient (vrp1Δ) cells are inviable at high temperature, have partially depolarized cortical actin patches and have defects in both actomyosin ring-dependent and Hof1 (Cyk2)-dependent pathways of cytokinesis. We demonstrate here that N-Vrp11–364 and C-Vrp1364–817 are each sufficient to restore viability, actomyosin ring constriction and Hof1 localization at 37°C to vrp1Δ. C-Vrp1, like Vrp1, partially co-localizes with cortical actin patches and restores actin patch polarization to vrp1Δ. Cortical localization of C-Vrp1, but not Vrp1, requires Las17. N-Vrp1 exhibits diffuse cytoplasmic localization and functions in cytokinesis without efficiently restoring polarization of cortical actin patches. N-Vrp1 function is not abolished by mutations affecting the WASP homology 2 (WH2) [verprolin homology (V)] actin-binding domain. N-Vrp1 may function through the type I myosins and actin, while C-Vrp1 may function through both Las17 (Bee1) and type I myosins. The functions of Vrp1 in viability at 37°C and cytokinesis do not require efficient localization to, and function in, the cortical actin cytoskeleton. Introduction In Saccharomyces cerevisiae, the cortical actin cytoskeleton takes the form of F-actin patches and these undergo characteristic changes in distribution through the cell cycle. Actin patches polarize into small emerging buds, are randomly dispersed in the mother and large bud during mitosis, and upon exit from mitosis repolarize to the neck region where they are thought to function in cytokinesis (Adams and Pringle, 1984; Lew and Reed, 1993; reviewed in Winsor and Schiebel, 1997). In addition, F-actin is recruited into a type II myosin (Myo1p)-containing ring at the bud neck in late anaphase. This actomyosin ring undergoes a contraction-like event during cytokinesis and then disappears (Bi et al., 1998; Lippincott and Li, 1998a). Vrp1p is an S.cerevisiae proline-rich actin-associated protein related to mammalian Wiskott–Aldrich syndrome protein (WASP)-interacting protein (WIP) (Donnelly et al., 1993; Vaduva et al., 1997; 1999; Naqvi et al., 1998). Loss of Vrp1p leads to a partial loss of cortical actin patch polarization during the cell cycle (Donnelly et al., 1993). Moreover, Vrp1p localizes to cortical patches which partially co-localize with cortical actin patches (Vaduva et al., 1997; Naqvi et al., 1998). Vrp1p interacts with actin through residues 1–70 which contain a WASP homology 2 (WH2)/verprolin homology (V) domain including a single actin-binding motif (KLKKAET) (Vaduva et al., 1997). WH2/V domains are also found in WIP and other mammalian actin monomer-binding proteins such as members of the WASP/Scar/WAVE family (Vaduva et al., 1999; Welch, 1999; Higgs and Pollard, 2001; Martinez-Quiles et al., 2001). The WH2/V domain in WASP/Scar/WAVE family proteins is required for activation of the Arp2/3p complex (Machesky and Insall, 1998). Mutation of the KLKKAET motif in Vrp1p abolishes interaction with actin, and deletion of residues 1–70 has been reported to abolish function (Vaduva et al., 1997). Similarly, the actin-binding motif of human WIP has been reported to be essential for complementation of vrp1-1 (Vaduva et al., 1999). The C-terminal fragment of Vrp1p interacts with Las17p, the S.cerevisiae homologue of mammalian WASP (Naqvi et al., 1998; Madania et al., 1999; Evangelista et al., 2000), while multiple fragments of Vrp1p have been shown to interact with the SH3 domains of type I myosins Myo3p and Myo5p (Anderson et al., 1998; Evangelista et al., 2000). Both Las17p and the type I myosins localize to cortical patches which partially co-localize with cortical actin patches during the cell cycle, and localization of type I myosins has been shown to be dependent on Vrp1p (Goodson et al., 1996; Li, 1997; Anderson et al., 1998). Both Las17p and type I myosins have a general role in actin patch polarization (Goodson et al., 1996; Karpova et al., 1998) and actin filament assembly through activation of the yeast Arp2/3p complex (Winter et al., 1999; Evangelista et al., 2000; Geli et al., 2000; Lechler et al., 2000). Type I myosins, unlike Las17p, lack a WH2/V domain, and Vrp1p may thus facilitate type I myosin function by supplying actin monomers (Geli et al., 2000). Vrp1p-deficient cells (vrp1Δ) are viable at 24°C, but not at 37°C (Donnelly et al., 1993), and loss of viability at 37°C is associated with a tight block in cytokinesis and defects in both actomyosin ring constriction and localization of the cytokinesis-specific Hof1p protein to a single ring at the bud neck (Naqvi et al., 2001). Hof1p is the S.cerevisiae homologue of Schizosaccharomyces pombe Cdc15p and is related to the mammalian cleavage furrow protein, proline serine threonine phosphatase-interacting protein (PSTPIP) (Kamei et al., 1998; Lippincott and Li, 1998b; Vallen et al., 2000). Cell division in S.cerevisiae does not absolutely require the function of the Myo1p-containing actomyosin ring (Bi et al., 1998; Lippincott and Li, 1998a). In the absence of an actomyosin ring (e.g. in myo1Δ) or when actomyosin ring constriction is prevented (e.g. in bni1Δ), another pathway of cytokinesis, which is redundant with the Myo1p pathway and probably involves directed secretion to the bud neck, can mediate septation. This alternative pathway relies on Hof1p function, since loss of both actomyosin ring function and Hof1p function (e.g. in myo1Δ hof1Δ or bni1Δ hof1Δ double mutants) leads to inviability (Vallen et al., 2000). The independence of these two pathways is indicated by the fact that Hof1p relocalization to a single ring at the bud neck does not require Myo1p and that Myo1p ring constriction does not require Hof1p (Vallen et al., 2000). The subcellular localization of Vrp1p and its role in cortical actin patch polarization suggest that its vital functions may be in cortical actin patches (Vaduva et al., 1997; Smith et al., 2001). Defective cytokinesis may be a consequence of aberrant actin patch polarization. The possibility remains, however, that the functions of Vrp1p in cytokinesis and in cortical actin patches are independent. Here we have addressed this question by demonstrating that both N-Vrp1p1–364 and C-Vrp1p364–817 are sufficient to replace Vrp1p in actomyosin ring constriction and Hof1p localization and, moreover, that N-Vrp1p functions in cytokinesis without either efficiently localizing to cortical actin patches nor fully restoring their polarization. C-Vrp1p localizes to the cortex and restores full cortical actin patch polarization, and localization requires Las17p. The localization and various functions of Vrp1p (and the cytokinesis functions of N-Vrp1p) do not require the WH2/V domain. Both Las17p and type I myosins are important for Hof1p localization to the bud neck during cytokinesis, suggesting a possible general requirement for Arp2/3p-dependent actin polymerization in this process. Results N- and C-terminal fragments of Vrp1p can substitute for full-length Vrp1p in restoration of viability at 37°C During mutational analysis of plasmid-borne VRP1, we noticed that all six mutations we isolated which affect Vrp1p function cause premature chain termination and map to the first 120 codons of VRP1. Amino acid substitution mutations, which were also present in most of these mutagenized plasmids, did not confer phenotypes after separation from the terminator mutations. Further more, extensive site-directed mutagenesis of charged amino acid clusters in plasmid-borne VRP1 did not yield noticeable phenotypes (data not shown). Interestingly, subsequent testing of the ability of individual fragments of Vrp1p to rescue the defects of vrp1Δ revealed that the N-terminal (1–364) (N-Vrp1p) and C-terminal (364–817) (C-Vrp1p) fragments are each functional. Centromeric plasmids expressing either N-Vrp1p or C-Vrp1p from the endogenous VRP1 promoter, but not empty vector, were each able to improve viability at 24°C significantly and restore viability at 37°C when introduced into vrp1Δ. Restoration of viability by N-Vrp1p and C-Vrp1p was observed both on solid (Figure 1A) and in liquid (Figure 1B) media. C-Vrp1p was significantly more efficient than N-Vrp1p in each case and restored a doubling time to vrp1Δ comparable with that of full-length Vrp1p (Figure 1B). Figure 1.Both N-Vrp1p and C-Vrp1p restore viability to vrp1Δ. (A) Viability at 24 and 37°C of vrp1Δ (AMY88) carrying YCplac111 vector (Vect), pAM228 (Vrp1p), pAM232 (N-Vrp1p) and pAM236 (C-Vrp1p). Each strain was streaked for single colonies on YPUAD solid medium, incubated at either 24 or 37°C, and photographed after 3 days. (B) Growth rate of vrp1Δ (AMY88) carrying YCplac111 vector (Vect), pAM228 (Vrp1p), pAM232 (N-Vrp1p) or pAM236 (C-Vrp1p). A YPUAD culture of each strain was grown at 24°C, diluted to an OD600 of 0.05 in fresh YPUAD medium and incubated at either 24 or 37°C. The OD600 was monitored at 1 h intervals. Download figure Download PowerPoint The complementation of vrp1Δ by N-Vrp1p and C-Vrp1p is not due to the spontaneous appearance of suppressor mutations in our vrp1Δ strain. We observed strong complementation by N-Vrp1p or C-Vrp1p, but not by the empty vector, in many experiments involving the use of more than one vrp1Δ mutant strain. Complementation is also not dependent on the genetic background, since similar results were obtained using three other vrp1Δ strains: Y15246 (from Euroscarf, Frankfurt, Germany), T65-1D (Zoladek et al., 1995) and vrp1Δ derivatives of YEF1986 (Vallen et al., 2000) and YEF1698 (Bi et al., 1998) constructed in our laboratory (data not shown). Further analysis revealed that C-Vrp1p is able to restore full polarization of cortical actin patches to vrp1Δ, while N-Vrp1p restores viability without correcting this defect efficiently (Figure 2A, Table I). That N-Vrp1p is not able to correct the cytoskeletal defects of vrp1Δ efficiently was supported further by the observation that the sensitivity of vrp1Δ strains to 1 M NaCl at 24°C was not corrected by N-Vrp1p, although, in contrast, C-Vrp1p was able to correct this defect (Figure 2B). Figure 2.C-Vrp1p, but not N-Vrp1p, restores full polarization of cortical actin patches to vrp1Δ. (A) Cortical actin patch polarization of vrp1Δ (AMY88) carrying YCplac111 vector (Vect), pAM228 (Vrp1p), pAM232 (N-Vrp1p) or pAM236 (C-Vrp1p). Cells were grown in YPUAD to exponential phase at 24°C and then either left at 24°C or shifted to 37°C for 2 h. The cells were fixed, permeabilized and F-actin stained with Alexa-488-conjugated phalloidin. Stained cells were viewed by fluorescence microscopy. Fields containing small-budded cells are shown to compare the polarization of cortical actin patches at this stage of the cell cycle. All panels are at equal magnification. (B) Salt sensitivity of vrp1Δ (AMY88) carrying YCplac111 vector (Vect), pAM228 (Vrp1p), pAM232 (N-Vrp1p) or pAM236 (C-Vrp1p). Each strain was streaked for single colonies on YPUAD solid medium containing 1 M sodium chloride, incubated at 24°C and photographed after 5 days. (C) Total protein extracts from vrp1Δ (AMY88) carrying pAM249 (Vrp1p-Pk), pAM250 (N-Vrp1p-Pk) or pAM251 (C-Vrp1p-Pk) after growth to exponential phase in YPUAD at 24°C were resolved by SDS–PAGE, transferred to a PVDF membrane and immunoblotted with anti-Pk antisera (αPK). We also immunoblotted with α-hexokinase as a loading control (αHex). The band intensities were quantified and full-length Vrp1p-Pk was normalized to 1. C-Vrp1p-Pk was 3.5 while N-Vrp1p-Pk was 12.7. Download figure Download PowerPoint Table 1. Actin patch polarization in vrp1Δ cells carrying vector, or plasmids expressing various forms of Vrp1p Plasmids 24°C 37°C Fully polarized Depolarized Fully polarized Depolarized Vector 1 99 0 100 Vrp1p 94 6 90.5 9.5 Vrp1pKA 91.5 8.5 92.5 7.5 Vrp1pKE 93.5 6.5 91.5 8.5 Vrp1p69–817 92.5 7.5 91 9 C-Vrp1p 86.5 13.5 61.5 38.5 N-Vrp1p 17.5 82.5 19 81 N-Vrp1p-CAAX 37 63 41 59 The difference in functionality between N-Vrp1p and C-Vrp1p is not due to differences in expression level or protein stability. Equivalent plasmids expressing Pk epitope-tagged full-length Vrp1p, N-Vrp1p or C-Vrp1p were introduced into vrp1Δ. Immunoblotting of extracts showed that N-Vrp1p-Pk is significantly more abundant than C-Vrp1p-Pk, and that C-Vrp1p-Pk is slightly more abundant than full-length Vrp1p-Pk in vivo (Figure 2C). Similar results were obtained with green fluorescent protein (GFP)-tagged constructs (Figure 6B). N-Vrp1p and C-Vrp1p each function in cytokinesis by restoring actomyosin ring constriction and Hof1p relocalization to a single ring at 37°C We next tested the ability of the Vrp1p fragments to rescue the actomyosin ring constriction and Hof1p localization defects at 37°C of vrp1Δ (Naqvi et al., 2001). Plasmids expressing full-length Vrp1p, N-Vrp1p, C-Vrp1p or the empty vector were introduced into vrp1Δ strains expressing either a functional Myo1p–GFP fusion protein or a functional Hof1p–GFP fusion protein. The Myo1p–GFP and Hof1p–GFP fusion proteins were visualized by fluorescence microscopy in living cells after shifting to 37°C for 3 h (Figure 3A and B, respectively, and Table II). The majority of Myo1p–GFP rings appeared uncontracted at 37°C in vrp1Δ carrying empty vector, as previously reported (Naqvi et al., 2001). Myo1p–GFP ring contraction at 37°C was restored by expression of either N-Vrp1p, C-Vrp1p or full-length Vrp1p (Figure 3A, Table II). Full-length Vrp1p and C-Vrp1p were approximately equally efficient, and both were more efficient than N-Vrp1p (Table II). Figure 3.N-Vrp1p and C-Vrp1p each function in both actomyosin ring constriction and Hof1p localization to a single ring at the bud neck prior to cytokinesis. (A) Actomyosin ring constriction in vrp1Δ expressing a functional Myo1p–GFP fusion protein (AMY114) and carrying YCplac111 vector (Vect), pAM228 (Vrp1p), pAM232 (N-Vrp1p) or pAM236 (C-Vrp1p). Cells were grown in YPUAD to exponential phase at 24°C and shifted to 37°C for 3 h. GFP was visualized in living cells by fluorescence microscopy. The arrows point to constricted Myo1p–GFP rings present at the neck of large-budded cells. Upper panels: fluorescence optics. Lower panels: DIC optics. All panels are at equal magnification. (B) Hof1p localization to a single ring at the bud neck at 37°C in vrp1Δ expressing a functional Hof1p–GFP fusion protein (AMY147) and carrying YCplac111 vector (Vect), pAM228 (Vrp1p), pAM232 (N-Vrp1p) or pAM236 (C-Vrp1p). Cells were grown in YPUAD to exponential phase at 24°C and shifted to 37°C for 3 h. GFP was visualized in living cells by fluorescence microscopy. Arrows: Hof1p–GFP single rings present at the neck of large-budded cells. Upper panels: FITC fluorescence optics. Lower panels: DIC optics. All panels are at equal magnification. Download figure Download PowerPoint Table 2. Hof1p–GFP relocalization and Myo1p–GFP ring constriction in large-budded vrp1Δ cells carrying vector, or plasmids expressing Vrp1p, N-Vrp1p, C-Vrp1p or N-Vrp1p-CAAX Vector Vrp1p N-Vrp1p C-Vrp1p N-Vrp1p-CAAX Hof1p–GFP Single ring NDa 59 32 61 51 Double ring NDa 41 68 39 49 Myo1p–GFP Constricted 1 47.5 33 52 65.5 Unconstricted 99 52.5 67 48 34.5 aMost cells had diffuse cytoplasmic fluorescence after shifting to 37°C for 3 h. In this vrp1Δ strain (AMY147), we observed stronger Hof1p mislocalization than we previously reported for RH2892 (Naqvi et al., 2001) (Figure 3B). In AMY147, we found mainly diffuse Hof1p–GFP fluorescence in large-budded cells after 3 h at 37°C, and in only very few large-budded cells did we observe Hof1p–GFP in two rings at the bud neck. In agreement with our previous report (Naqvi et al., 2001), however, we observed no cells with Hof1p–GFP single rings under these conditions. Hof1p–GFP localization to double rings and subsequent relocalization to single rings at the bud neck was restored by expression of N-Vrp1p, C-Vrp1p or full-length Vrp1p, but not by empty vector (Figure 3B, Table II). Full-length Vrp1p and C-Vrp1p were approximately equally efficient, and both were more efficient than N-Vrp1p (Table II). The functions of N-Vrp1p in cytokinesis do not require the conserved WH2/V domain containing the actin-binding motif The finding that C-Vrp1p is fully functional suggests that the N-terminal actin-binding motif (KLKKAET) in the WH2/V domain may not be essential, in disagreement with a previous report (Vaduva et al., 1997). To re-examine the importance of the actin-binding motif, we made a construct expressing full-length Vrp1p in which K45 and K46 of the KLKKAET motif are substituted with alanine (Vrp1pKA) or glutamate (Vrp1pKE). Previously, substitution of K45 and K46 with glutamate was shown to abolish two-hybrid interaction between a fragment comprising residues 1–70 of Vrp1p and actin (Vaduva et al., 1997). We also detected a two-hybrid interaction between this fragment of Vrp1p and actin. Also consistent with the findings of Vaduva et al. (1997), this interaction was abolished by substitution of K45 and K46 (data not shown). Both Vrp1pKA and Vrp1pKE were analysed for their ability to restore viability (Figure 4A) and full polarization of cortical actin patches (Figure 4B) to vrp1Δ. Neither mutation significantly affected the function of Vrp1p in these processes. Previously, a mutant Gal4–Vrp1p fusion protein in which Vrp1p residues 1–70 were deleted was found to be unable to restore viability to vrp1-1 (Vaduva et al., 1997). We made a construct expressing residues 69–817 of Vrp1p (Vrp1p69−817) which is similar to the construct tested by Vaduva et al. (1997) except that in our construct VRP1 was not fused to GAL4. Vrp1p69–817 was as functional as full-length Vrp1p in restoring viability at 37°C and actin patch polarization to vrp1Δ (Figure 4A and B). Figure 4.The conserved WH2/V domain of Vrp1p is not essential for viability at 37°C or cortical actin patch polarization. (A) Viability at 24 and 37°C of vrp1Δ (AMY88) carrying YCplac111 vector (Vect), pAM228 (Vrp1p), pAM229 (Vrp1pKA), pAM230 (Vrp1pKE) or pAM231 (Vrp1p69–817). Each strain was streaked for single colonies on YPUAD solid medium, incubated at either 24 or 37°C and photographed after 3 days. (B) Cortical actin patch polarization in vrp1Δ (AMY88) carrying YCplac111 vector (Vect), pAM228 (Vrp1p), pAM229 (Vrp1pKA), pAM230 (Vrp1pKE) or pAM231 (Vrp1p69–817). Cells were grown in YPUAD to exponential phase at 24°C and either left at 24°C or shifted to 37°C for 2 h. The cells were fixed, permeabilized and F-actin stained with Alexa-488-conjugated phalloidin. Stained cells were viewed by fluorescence microscopy. Fields containing small-budded cells are shown to compare the polarization of cortical actin patches. All panels are at equal magnification. Download figure Download PowerPoint We next tested whether the KLKKAET actin-binding motif is essential for the functions of N-Vrp1p. Centro meric plasmids expressing forms of N-Vrp1p with the K45A K46A (N-Vrp1pKA) or K45E K46E (N-Vrp1pKE) actin-binding site mutations or with residues 1–68 deleted (Vrp1p69–364) were constructed and tested for their ability to restore viability to vrp1Δ at 24 and 37°C on solid medium (Figure 5A) and in liquid medium (Figure 5B). The K45A K46A and K45E K46E mutations did not significantly affect the function of N-Vrp1p. Deletion of residues 1–68 of N-Vrp1p, while not completely abolishing function, may have caused some loss of activity compared with the other mutations (Figure 5B). Thus, the WH2/V domain is not required, even for the function of the N-Vrp1p fragment. Figure 5.The conserved WH2/V domain is not essential for the function of N-Vrp1p in restoring viability at 37°C. (A) Viability at 24 and 37°C of vrp1Δ (AMY88) carrying YCplac111 vector (Vect), pAM232 (N-Vrp1p), pAM233 (N-Vrp1pKA), pAM234 (N-Vrp1pKE) or pAM235 (Vrp1p69–364). Each strain was streaked for single colonies on YPUAD solid medium, incubated at either 24 or 37°C and photographed after 3 days. (B) Growth rate of vrp1Δ (AMY88) carrying YCplac111 vector (Vect), pAM232 (N-Vrp1p), pAM233 (N-Vrp1pKA), pAM233 (N-Vrp1pKE) or pAM235 (Vrp1p69–364). A YPUAD culture of each strain was grown at 24°C, diluted to an OD600 of 0.05 in fresh YPUAD medium and incubated at 37°C. The OD600 was monitored at 1 h intervals. (C) Fragments of N-Vrp1p were tested for complementation of vrp1Δ (AMY88) and two-hybrid interaction with actin. Download figure Download PowerPoint We next carried out a more extensive deletion analysis on N-Vrp1p to identify sequences that are important for its function, as assayed by formation of colonies at 37°C (Figure 5C). Like deletion of residues 1–68, deletion of residues 270–364 did not abolish function, but may have reduced the activity of N-Vrp1p somewhat. A combination of both the 1–68 and 270–364 deletions, in contrast, did completely abrogate colony formation at 37°C. This result suggests that several regions of N-Vrp1p may have the ability to contribute, perhaps in similar ways, to the function of this fragment. C-Vrp1p localizes to cortical patches which partially co-localize with actin patches in a mechanism dependent on Las17p, while N-Vrp1p does not localize To investigate whether either fragment of Vrp1p contains sufficient information for localization to cortical patches, we tagged full-length Vrp1p, N-Vrp1p and C-Vrp1p with GFP and expressed them from the VRP1 promoter carried on a centromeric plasmid. As a control, we also made a construct expressing GFP under the control of the VRP1 promoter. The GFP-tagged Vrp1p fragments, but not GFP itself, were partially functional in complementation tests when expressed in vrp1Δ (data not shown). Live cell imaging showed that both full-length Vrp1p–GFP and C-Vrp1p–GFP localized predominantly to punctate cortical structures, while N-Vrp1p–GFP showed a diffuse cytoplasmic fluorescence pattern similar to that of GFP alone (Figure 6A). Lack of strong cortical localization of N-Vrp1p–GFP is not due to poor expression or instability of this fusion protein. Immunoblotting experiments showed that N-Vrp1p–GFP is expressed at somewhat higher levels than C-Vrp1p–GFP, and C-Vrp1p–GFP is expressed at somewhat higher levels than full-length Vrp1p–GFP (Figure 6B). Immunofluorescence localization revealed that C-Vrp1p–GFP, like full-length Vrp1p–GFP, partially co-localizes with cortical actin patches (Figure 6C). Next, we tested whether full-length Vrp1p–GFP or C-Vrp1p–GFP require Las17p for localization to cortical patches and for partial co-localization with cortical actin patches. Full-length Vrp1p–GFP localized efficiently to the cortex in Las17p-deficient cells (Figure 7A) and still partially co-localized with cortical F-actin patches, although these patches were delocalized (Figure 7B). In contrast, C-Vrp1p–GFP exhibited only diffuse cytoplasmic fluorescence similar to GFP alone (Figure 7A) in cells lacking Las17p. The region comprising the C-terminal 36 residues of Vrp1p interacts with Las17p (Madania et al., 1999). Deletion of this region (residues 760–817) in C-Vrp1p completely abolished its ability to complement vrp1Δ (data not shown). This suggests that the function of C-Vrp1p requires interaction with Las17p. Figure 6.C-Vrp1p localizes to cortical patches which partially co-localize with actin patches. (A) vrp1Δ (AMY88) carrying pAM237 (GFP), pAM162 (Vrp1p–GFP), pAM239 (N-Vrp1p–GFP) or pAM241 (C-Vrp1p–GFP) were grown in YPUAD to exponential phase at 24°C and GFP was visualized in living cells by fluorescence microscopy. Upper panels: FITC fluorescence optics. Lower panels: DIC optics. All panels are at equal magnification. (B) Total protein extracts from vrp1Δ (AMY88) carrying pAM237 (GFP), pAM162 (Vrp1p–GFP), pAM239 (N-Vrp1p–GFP) or pAM241 (C-Vrp1p–GFP) after growth to exponential phase in YPUAD at 24°C were resolved by SDS–PAGE, transferred to a PVDF membrane and immunoblotted with anti-GFP antisera (αGFP). We also immunoblotted with α-hexokinase as a loading control (αHex). (C) vrp1Δ (AMY88) carrying pAM162 (Vrp1p–GFP) or pAM241 (C-Vrp1p–GFP) were grown in YPUAD to exponential phase at 24°C, fixed, permeabilized and GFP was visualized by immunofluorescence (GFP). Cortical actin patches were visualized in the same cells by staining with Alexa-488-conjugated phalloidin (Actin). Stained cells were viewed by fluorescence microscopy using FITC- and rhodamine-specific light filters. Arrowheads: Vrp1p–GFP and C-Vrp1p–GFP patches which co-localize with cortical actin patches. All panels are at equal magnification. Download figure Download PowerPoint Figure 7.Localization of C-Vrp1p, but not full-length Vrp1p, to cortical patches requires Las17p. (A) las17Δ (IDY166) carrying pAM237 (GFP), pAM162 (Vrp1p–GFP) or pAM241 (C-Vrp1p–GFP) were grown in YPUAD to exponential phase at 24°C and GFP fluorescence was visualized in living cells by fluorescence microscopy. Upper panels: FITC fluorescence optics. Lower panels: DIC optics. All panels are at equal magnification. (B) las17Δ (IDY166) carrying pAM162 (Vrp1p–GFP) was grown in YPUAD to exponential phase at 24°C, fixed, permeabilized and GFP was visualized by immunofluorescence (GFP). Cortical actin patches were visualized in the same cells by staining with Alexa-488-conjugated phalloidin (Actin). Stained cells were viewed by fluorescence microscopy. Arrowheads: Vrp1p–GFP patches which co-localize with cortical actin patches. All panels are at equal magnification. Download figure Download PowerPoint N-Vrp1p-CAAX localizes to the cortex and exhibits improved function in cytokinesis We asked whether by artificially directing N-Vrp1p to the cortex we would restore any additional Vrp1p-dependent functions. Addition of a CAAX box to the C-terminus has been used to confer covalent lipid modification and strong cortical localization to proteins which do not normally localize to the cortex (Golsteyn et al., 1997). We constructed centromeric plasmids expressing from the VRP1 promoter forms of N-Vrp1p in which the C-terminus of the fragment was modified by addition of either the CAAX box of S.cerevisiae Ras1p (Powers et al., 1984) (N-Vrp1p-CAAX) or both GFP and the CAAX box (N-Vrp1p–GFP-CAAX). The Ras1p CAAX box was functional in these experiments since it conferred efficient cortical localization to GFP (Figure 8A). N-Vrp1p–GFP-CAAX was able to localize to the cortex, although the distribution was diffuse at the cortex without predominant patches, similar to GFP–CAAX (Figure 8A). N-Vrp1p-CAAX was able to restore growth at 37°C on solid media (Figure 8B) and in liquid media (Figure 8C) better than N-Vrp1p when expressed in vrp1Δ. N-Vrp1p-CAAX was approximately as functional in these assays as either C-Vrp1p or full-length Vrp1p (Figure 1A and B). The improved growth at 37°C correlated with improved ability of N-Vrp1p-CAAX, compared with N-Vrp1p alone, to restore actomyosin ring constriction and Hof1p–GFP localization to single rings at the bud neck during cytokinesis (Table II). Interestingly, the improved function of N-Vrp1p-CAAX in cytokinesis was not associated with full restoration of cortical actin patch polarization, although some improvement
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