Intracellular localization of Arabidopsis sulfurtransferases

Abstract

Sulfurtransferases (Str) comprise a group of enzymes widely distributed in archaea, eubacteria, and eukaryota which catalyze the transfer of a sulfur atom from suitable sulfur donors to nucleophilic sulfur acceptors. In all organisms analyzed to date, small gene families encoding Str proteins have been identified. The gene products were localized to different compartments of the cells. Our interest concerns the localization of Str proteins encoded in the nuclear genome of Arabidopsis. Computer-based prediction methods revealed localization in different compartments of the cell for six putative AtStrs. Several methods were used to determine the localization of the AtStr proteins experimentally. For AtStr1, a mitochondrial localization was demonstrated by immunodetection in the proteome of isolated mitochondria resolved by one- and two-dimensional gel electrophoresis and subsequent blotting. The respective mature AtStr1 protein was identified by mass spectrometry sequencing. The same result was obtained by transient expression of fusion constructs with the green fluorescent protein in Arabidopsis protoplasts, whereas AtStr2 was exclusively localized to the cytoplasm by this method. Three members of the single-domain AtStr were localized in the chloroplasts as demonstrated by transient expression of green fluorescent protein fusions in protoplasts and stomata, whereas the single-domain AtStr18 was shown to be cytoplasmic. The remarkable subcellular distribution of AtStr15 was additionally analyzed by transmission electron immunomicroscopy using a monospecific antibody against green fluorescent protein, indicating an attachment to the thylakoid membrane. The knowledge of the intracellular localization of the members of this multiprotein family will help elucidate their specific functions in the organism.

Figure 1.

Subcellular localization of AtStr1 and AtStr2 GFP fusion constructs with and without signal peptide. A, Arabidopsis protoplasts were transformed with AtStr1 including its targeting peptide sequence (AtStr1wPS). Fluorescence images of the protoplasts were taken using a confocal laser scanning microscope. The GFP fluorescence was excited with the argon laser (488 nm) and detected at 515 nm to 520 nm. B, The same protoplast suspension was additionally stained with MitoTracker Orange CMTMRos. The fluorescence was excited with the green helium neon laser (543 nm) and detected at 575 nm to 585 nm. C–J, Arabidopsis protoplasts were transiently transformed with AtStr1 and AtStr2 constructs with and without the targeting peptide sequences (AtStr1wPS/AtStr1woPS and AtStr2wPS/AtStr2woPS, respectively). Bright field images shown in C, E, G, and I were made to visualize the protoplast's cell membrane and the chloroplasts. Fluorescence images of the protoplasts shown in D, F, H, and J were taken using an Axioskop microscope with filter sets optimal for GFP fluorescence (BP 450–490/LP 520). All scale bars represent 10 μm.

Figure 2.

Protein gel electrophoresis and subsequent western-blot analysis of total and organellar extracts. A, Mitochondria (Mi) were purified from Arabidopsis cell cultures, chloroplasts (Cp) were isolated from green Arabidopsis plants, and total soluble protein extracts (Te) were also obtained from green Arabidopsis plants. The proteins were separated by one-dimensional gel electrophoresis, blotted, and the membranes were incubated with an antibody directed against the AtStr1 protein. B, Mitochondria were purified as described above. Their proteome was separated by two-dimensional gel electrophoresis and analyzed by western-blot analysis as described above (top). The corresponding protein spots marked by arrows were localized on a Coomassie-stained gel that was run in parallel. The spots were cut and the proteins were analyzed by mass spectrometry.

Figure 3.

Localization of the single-domain Str AtStr14 and AtStr18 in tobacco leaf epidermal and guard cells. Fusion constructs of AtStr14 and AtStr18 with the GFP encoding sequence were transformed in N. tabacum leaf cells by particle gun bombardment and were analyzed by fluorescence microscopy after overnight incubation. A, Bright field image of an epidermal N. tabacum cell. B, In the same cell shown in A, the fluorescence of the AtStr14/pGFP-N fusion protein was collected with the band pass filter (BP 450–490) for excitation and with the long pass filter (filter LP 520) for emission. C, Single merged image of a guard cell for AtStr14. D, Bright field image of a guard cell. E, In the same guard cell shown in D, the fluorescence was collected as described in B. All scale bars represent 10 μm.

Figure 4.

Targeting analysis of all members of the AtStr group VI visualized by fluorescence microscopy. The fusion constructs of AtStr14, AtStr15, AtStr16, and AtStr18 with pGFP-N were introduced into Arabidopsis protoplasts. The protoplasts were incubated overnight at room temperature and then analyzed with an Axioskop microscope with filter sets optimal for GFP fluorescence (BP 450–490/LP 520). Bright field images (A, C, E, and G) were made to visualize the protoplast's cell membrane and chloroplasts. Fluorescence images of the same protoplasts are shown in B, D, F, and H.

Figure 5.

Detailed analysis of the subcellular localization of AtStr15. Arabidopsis protoplasts were transformed with the AtStr15/pGFP-N fusion construct. All images (A–F) were made with the True Confocal Scanner. GFP fluorescence was excited with an argon laser (488 nm) and detected at 515 nm to 520 nm. Chlorophyll autofluorescence (red) was detected simultaneously at 650 nm to 670 nm. A, GFP fluorescence in a transformed protoplast. B, chlorophyll fluorescence of the same protoplast as in A. C, Merged image of A and B. D, This figure represents the image shown in C having coordinating lines to show the localization of AtStr15 (shown on the sidelines). E, GFP fluorescence of a single chloroplast merged with the respective bright field image. F, Enlarged image of the inset of Figure E. The sizes of the scale bars are given directly in the images.

Figure 6.

Immunogold localization of GFP fusion protein in transiently transformed protoplasts by transmission electron microscopy. A, Chloroplast with numerous (>12) gold particles (i.e. GFP immunoreactivity) close to another chloroplast with and to a mitochondrion (below) without immunogold label. B, Chloroplast at the left with about 12 gold particles at the thylakoid membrane. The mitochondrion at the right has no label. The chloroplast at the right contains only one label at the thylakoid membrane. The plasma membrane of the protoplast has one gold label. The scale bars represent 500 nm.