A novel amphiphilic motif at the C-terminus of FtsZ1 facilitates chloroplast division

Abstract

In bacteria and chloroplasts, the GTPase filamentous temperature-sensitive Z (FtsZ) is essential for division and polymerizes to form rings that mark the division site. Plants contain two FtsZ subfamilies (FtsZ1 and FtsZ2) with different assembly dynamics. FtsZ1 lacks the C-terminal domain of a typical FtsZ protein. Here, we show that the conserved short motif FtsZ1 Carboxyl-terminus (Z1C) (consisting of the amino acids RRLFF) with weak membrane-binding activity is present at the C-terminus of FtsZ1 in angiosperms. For a polymer-forming protein such as FtsZ, this activity is strong enough for membrane tethering. Arabidopsis thaliana plants with mutated Z1C motifs contained heterogeneously sized chloroplasts and parallel FtsZ rings or long FtsZ filaments, suggesting that the Z1C motif plays an important role in regulating FtsZ ring dynamics. Our findings uncover a type of amphiphilic beta-strand motif with weak membrane-binding activity and point to the importance of this motif for the dynamic regulation of protein complex formation.

A Tissue-Chopping Based Immunofluorescence Staining Method for Chloroplast Proteins

“…The sample was covered with 1 mL blocking solution (5% BSA in 1 × PBS with 0.15% Triton X-100) and incubated at room temperature for 30 min. The samples were then incubated with 200 μL anti-FtsZ1 antibodies ( Liu et al, 2022 ) (a dilution of 1:100 with blocking solution) for 2 h in darkness at room temperature. Next, samples were washed with 1 × PBS or 5% BSA in 1 × PBS three times.…”

“…Immunofluorescence staining is a widely used method to study the localization of proteins in the cell. It is frequently used to study the localization of chloroplast division proteins ( Vitha et al, 2001 ; Li et al, 2016 ; Wang et al, 2017 ; Liu et al, 2022 ). Compared with transforming plants with fluorescent protein tags, immunofluorescence staining does not need to obtain transgenic plants, so it is a good choice to study the localization of proteins in plants that cannot be transformed or are untransformed.…”

“…In comparison with the tissue sectioning method, a new method using isolated protoplasts for the immunofluorescence staining of chloroplast proteins was developed ( Li et al, 2016 ). This method was used in studying the localization of chloroplast division proteins ( Wang et al, 2017 ; Liu et al, 2022 ). Compared with the immunofluorescence staining method with wax-embedded tissue sections, this method with enzymatic digestion of cell wall is much simpler and less time-consuming.…”

Improvements for Tissue-Chopping-Based Immunofluorescence Staining Method of Chloroplast Proteins

“…Immunofluorescence staining is a frequently used method in the field of cell biology. Immunofluorescence staining is often used to observe the localization of chloroplast division proteins [ 8 , 9 , 10 , 11 , 12 ]. For plants that are difficult to obtain transgenic materials for or wild samples collected in natural habitats, immunofluorescence staining is the best choice to study the localization of proteins.…”

“…With this method, leaf tissue was subjected to enzymatic digestion first, and then the immunofluorescence staining was performed with protoplasts [ 9 ]. This method has been widely used in the localization study of chloroplast division proteins [ 10 , 11 ]. Compared with the sectioning-based immunofluorescence staining method, enzymatic digestion is much simpler and less time-consuming, and the results can be obtained in only one day.…”