![Figure 4. Inn1–Hof1 interaction and its role in the symmetric localization of Inn1 at the neck. (A) Coimmunoprecipitation and cell cycle–dependent modification of Inn1 and Hof1. Strain MOY157 (INN1-GFP HOF-TAP cdc15-2) was grown to exponential phase in YM-P medium at 24°C, shifted to 37°C for 2.5 h to synchronize cells at mitotic exit using the cdc15-2 block, released to permissive temperature by rapidly cooling to 24°C, and sampled at intervals. Hof1-TAP was precipitated from protein extracts, and samples of the extracts (input) and precipitates (IP) were analyzed by SDS-PAGE and immunoblotting. In a control in which no TAP-tagged protein was present, no Inn1-GFP was detected in the precipitate (not depicted). (B) Phosphorylation of Inn1. Strain MOY215 (INN1-GFP cdc15-2) was synchronized as in A and sampled 45 min after release. Inn1-GFP was immunoprecipitated and subjected to phosphatase treatments as indicated. (C) Two-hybrid analysis of Inn1–Hof1 interaction. The diagram shows the domain structures of Inn1 (m1-m4 are the mutations introduced into the PXXP motifs; see Results; Fig. S1) and Hof1. FCH, FER/CIP4 homology; CC, coiled coil; SH3, Src homology 3. Various Inn1 fragments carried on the AD (AD-Vect) were tested pairwise for interaction with full-length Hof1 (Hof1-FL), Hof1 amino acids 576–669 (Hof1-SH3), and Hof1 amino acids 1–340 (Hof1–F-BAR) carried on the DBD vector (DBD-Vect). Asterisk indicates that Inn1(1–180) interacted with Hof1-SH3 for unknown reasons. (D) Role of Inn1 amino acids 377–383 (PXXPPXP) in the Inn1–Hof1 interaction. Two-hybrid analysis was conducted using full-length Hof1-DBD and Inn1(131–409)-AD. The Inn1 sequence was wild type (tail[131–409]) or carried mutations m1, m2, m3, and/or m4 individually or in combinations. (E) Direct binding of Inn1 to Hof1 and its mediation by Inn1 amino acids 377–383 (PKLPPLP). Purified GST-Inn1-tail (amino acids 131–409; wild type [WT] or carrying mutation m2 or m4) and His6-Hof1-C (amino acids 341–669) were tested for binding in vitro as described in Materials and methods. (F) Asymmetric localization of Inn1 at the neck in hof1Δ cells. Strain LY1328 (INN1-GFP hof1Δ [pRS316-HOF1]) was transformed with plasmid YCp111-CDC3-CFP and incubated on an FOA plate to eliminate the HOF1 plasmid. Cells from a population growing exponentially in SC-Leu medium at 24°C were examined by 3D microscopy (see Materials and methods). 1DC, one central dot (as typically observed in wild-type cells); 1DA, one asymmetric dot (as often observed in hof1Δ cells; Table I; and Videos 5 and 6). Bar, 2 µm.](https://cdn.rupress.org/rup/content_public/journal/jcb/185/6/10.1083_jcb.200903125/7/jcb_200903125_rgb_fig4.jpeg?Expires=1773794424&Signature=jA5wLobZFOwrPb055jXq1jPRh3FTFpiv6lwfTgSEbuXYWuRc45And-4y8w6pLCkX6KLveuBBONPDUNC-SC0kr2t~o-HeTMfXqx4FDTbCcuOsd~348391eBDzcnl8r2qsQAuzj6JGToRPXs-23Uk0LCwNx03AnEXAJnIP~gCuD9qVq9wUQlru2HtR-Y-1VgnSq7IwPgp~qrTKGFasdwnsylUM4zsKnWtdVpVjkZA1cDu3AZp3ChDBu~gC7xB0k8O-2dpMQs5x8AKkZpkdiCkYo8-sGUsQxQwrcECZBsMCO4P0uGnSdC922qdiXawf4J-mQdhyeVFpvGNHYfFwwA154w__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Inn1–Hof1 interaction and its role in the symmetric localization of Inn1 at the neck. (A) Coimmunoprecipitation and cell cycle–dependent modification of Inn1 and Hof1. Strain MOY157 (INN1-GFP HOF-TAP cdc15-2) was grown to exponential phase in YM-P medium at 24°C, shifted to 37°C for 2.5 h to synchronize cells at mitotic exit using the cdc15-2 block, released to permissive temperature by rapidly cooling to 24°C, and sampled at intervals. Hof1-TAP was precipitated from protein extracts, and samples of the extracts (input) and precipitates (IP) were analyzed by SDS-PAGE and immunoblotting. In a control in which no TAP-tagged protein was present, no Inn1-GFP was detected in the precipitate (not depicted). (B) Phosphorylation of Inn1. Strain MOY215 (INN1-GFP cdc15-2) was synchronized as in A and sampled 45 min after release. Inn1-GFP was immunoprecipitated and subjected to phosphatase treatments as indicated. (C) Two-hybrid analysis of Inn1–Hof1 interaction. The diagram shows the domain structures of Inn1 (m1-m4 are the mutations introduced into the PXXP motifs; see Results; Fig. S1) and Hof1. FCH, FER/CIP4 homology; CC, coiled coil; SH3, Src homology 3. Various Inn1 fragments carried on the AD (AD-Vect) were tested pairwise for interaction with full-length Hof1 (Hof1-FL), Hof1 amino acids 576–669 (Hof1-SH3), and Hof1 amino acids 1–340 (Hof1–F-BAR) carried on the DBD vector (DBD-Vect). Asterisk indicates that Inn1(1–180) interacted with Hof1-SH3 for unknown reasons. (D) Role of Inn1 amino acids 377–383 (PXXPPXP) in the Inn1–Hof1 interaction. Two-hybrid analysis was conducted using full-length Hof1-DBD and Inn1(131–409)-AD. The Inn1 sequence was wild type (tail[131–409]) or carried mutations m1, m2, m3, and/or m4 individually or in combinations. (E) Direct binding of Inn1 to Hof1 and its mediation by Inn1 amino acids 377–383 (PKLPPLP). Purified GST-Inn1-tail (amino acids 131–409; wild type [WT] or carrying mutation m2 or m4) and His6-Hof1-C (amino acids 341–669) were tested for binding in vitro as described in Materials and methods. (F) Asymmetric localization of Inn1 at the neck in hof1Δ cells. Strain LY1328 (INN1-GFP hof1Δ [pRS316-HOF1]) was transformed with plasmid YCp111-CDC3-CFP and incubated on an FOA plate to eliminate the HOF1 plasmid. Cells from a population growing exponentially in SC-Leu medium at 24°C were examined by 3D microscopy (see Materials and methods). 1DC, one central dot (as typically observed in wild-type cells); 1DA, one asymmetric dot (as often observed in hof1Δ cells; Table I; and Videos 5 and 6). Bar, 2 µm.
