1. RISAP Is a Peripheral Membrane Protein That Interacts with F-Actin
RISAP Is a Peripheral Membrane Protein That Interacts with F-Actin.To investigate RISAP membrane association and F-actin binding, extracts of normally growing stably transformed tobacco (SR1YFP:RISAP) pollen tubes displaying subapical YFP:RISAP accumulation...
RISAP Is a Peripheral Membrane Protein That Interacts with F-Actin.To investigate RISAP membrane association and F-actin binding, extracts of normally growing stably transformed tobacco (SR1YFP:RISAP) pollen tubes displaying subapical YFP:RISAP accumulation were fractionated using different high-speed centrifugation techniques. Fractions obtained were analyzed by immunoblotting using the indicated antibodies including a polyclonal peptide antibody directed against the RISAP C terminus (α-RISAP) ([A], [B], [D], and [E]). In addition, actin coimmunoprecipitation from wild-type tobacco pollen tube extracts together with myc-tagged RISAP was investigated (C).(A) YFP:RISAP partitioning between supernatant (S10) and pellet (P10) after centrifugation of regular crude SR1YFP:RISAP extract (CE) at 10,000g.(B) Partitioning of YFP:RISAP, actin, and the soluble protein RHOGDI2 between supernatant (S10) and pellet (P10) after 10,000g centrifugation of a crude SR1YFP:RISAP extract (CE) prepared in high ionic strength CDB buffer, which destabilizes F-actin (left column), or in low ionic strength CSB buffer, which promotes actin polymerization (right column).(C) In vitro-transcribed/translated myc-tagged RISAP was incubated in a wild-type tobacco pollen tube extract and immunoprecipitated using an α-myc antibody coupled to beads. Proteins associated with washed beads (α-myc CoIP), as well as myc:RISAP levels in the in vitro transcription/translation reaction mix and actin levels in pollen tube extracts (input), were analyzed by immunoblotting using α-myc (top panel) or α-actin antibodies (bottom panel). myc:RISAP free in vitro transcription/translation reaction mix added to pollen tube extract was used as a control.(D) S10 supernatant obtained by centrifugation of crude SR1YFP:RISAP extract at 10,000g was centrifuged at 150,000g to separate membrane-associated proteins in microsomal pellets from soluble proteins in the supernatant. Microsomal pellets were resuspended in buffer containing Triton X-100, SDS, NaCl, urea, or Na2CO3 at the indicated concentrations to solubilize different classes of membrane proteins. After incubation, resuspended microsomal pellets were centrifuged again at 150,000g. RISAP (top panel) and actin (bottom panel) partitioning between supernatants (S) and pellets (P) was analyzed by immunoblotting.(E) S10 supernatant obtained by centrifugation of crude SR1YFP:RISAP extract at 10,000g was adjusted to 80% sucrose and sequentially overlaid with two layers of buffer containing 65 and 10% sucrose, respectively. After centrifugation at 100,000g, nine fractions were collected starting at top of the gradient. The presence of RISAP as well as of actin in each of these fractions was analyzed by immunoblotting and compared with the distribution patterns of a soluble protein (RHOGDI2) and of two membrane-associated proteins (RAC5 and ARF1).
Octavian Stephan et al. Plant Cell 2014;26:
©2014 by American Society of Plant Biologists