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STIC2 selectively binds ribosome-nascent chain complexes in the cotranslational sorting of Arabidopsis thylakoid proteins

ABSTRACT: Chloroplast-encoded multi-span thylakoid membrane proteins are crucial for photosynthetic complexes, yet the coordination of their biogenesis remains poorly understood. To identify factors that specifically support the cotranslational biogenesis of the reaction center protein D1 of photosystem (PS) II, we generated and affinity-purified stalled ribosome-nascent chain complexes (RNCs) bearing D1 nascent chains. Stalled RNCs translating the soluble ribosomal subunit uS2c were used for comparison. Quantitative tandem-mass spectrometry of the purified RNCs identified around 140 proteins specifically associated with D1 RNCs, mainly involved in protein and cofactor biogenesis, including chlorophyll biosynthesis, and other metabolic pathways. Functional analysis of STIC2, a newly identified D1 RNC interactor, revealed its cooperation with chloroplast protein SRP54 in the de novo biogenesis and repair of D1, and potentially other cotranslationally-targeted reaction center subunits of PSII and PSI. The primary binding interface between STIC2 and the thylakoid insertase Alb3 and its homolog Alb4 was mapped to STIC2’s β-sheet region, and the conserved Motif III in the C-terminal regions of Alb3/4.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

TISSUE(S): Plant Cell, Leaf

SUBMITTER: Jan Lambertz

LAB HEAD: Marc M.

PROVIDER: PXD042896 | Pride | 2024-08-28

REPOSITORIES: Pride

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			Action	DRS
	2021_10_06_DS_Pisum_Database_BASIC.fasta	Fasta
	2021_10_06_DS_Pisum_Database_TS-Tags.fasta	Fasta
	JL_DS_D1_056_1_A.raw	Raw
	JL_DS_D1_056_1_B.raw	Raw
	JL_DS_D1_056_2_A.raw	Raw

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Project description:The cyclin D1 oncogene encodes the regulatory subunit of a holoenzyme that phosphorylates and inactivates the Rb protein and promotes progression through G1 to S phase of the cell cycle. Several prostate cancer cell lines and a subset of primary prostate cancer samples have increased cyclin D1 protein expression. However, the relationship between cyclin D1 expression and prostate tumor progression has yet to be clearly characterized. This study examined the effects of manipulating cyclin D1 expression in either human prostatic epithelial or stromal cells using a tissue recombination model. The data showed that overexpression of cyclin D1 in the initiated BPH-1 cell line increased cell proliferation rate, but did not elicit tumorigenicity in vivo. However, overexpression of cyclin D1 in Normal Prostate Fibroblasts (NPF) that were subsequently recombined with BPH-1 did induce malignant transformation of the epithelial cells. The present study also showed that recombination of BPH-1 + cyclin D1 overexpressing fibroblasts (NPF cyclin D1) resulted in permanent malignant transformation of epithelial cells (BPH-1 NPF-cyclin D1 cells) similar to that seen with Carcinoma Associated Fibroblasts (CAFs). Microarray analysis showed that the expression profiles between CAFs and NPF cyclin D1 cells were highly concordant including cyclin D1 upregulation. These data indicated that the tumor-promoting activity of cyclin D1 may be tissue-specific. Keywords: cyclin D1; stromal-epithelial interactions; prostate cancer; cDNA microarray NPF cyclin D1 cells were generated from NPFs, which were isolated from two different patient samples; CAFs were isolated from two different patient samples as well. RNA was isolated from NPFs, CAFs, and NPF cyclin D1 cells using total RNA isolation kit (Qiagen). Custom spotted cDNA microarrays were constructed as previously described (True, L., Coleman, I., Hawley, S., Huang, C. Y., Gifford, D., Coleman, R., Beer, T. M., Gelmann, E., Datta, M., Mostaghel, E., Knudsen, B., Lange, P., Vessella, R., Lin, D., Hood, L., and Nelson, P. S. A molecular correlate to the Gleason grading system for prostate adenocarcinoma. Proc Natl Acad Sci U S A, 103: 10991-10996, 2006) using a non-redundant set of 6,700 prostate-derived cDNA clones identified from the Prostate Expression Data Base (PEDB), a public sequence repository of expressed sequence tag data derived from human prostate cDNA libraries. Total RNA was amplified through one round of linear amplification using the MessageAmp aRNA kit (Ambion, Austin, TX). Sample quality and quantification was assessed by agarose gel electrophoresis and absorbance at A260. Cy3 and Cy5 labeled cDNA probes were made from 4 M-BM-5g of amplified RNA. Two NPF cyclin D1 and two CAF samples (labeled with Cy3) were hybridized head-to-head with an NPF control sample labeled with Cy5. Probes were hybridized competitively to microarrays under a coverslip for 16 h at 63M-BM-0C. Fluorescent array images were collected for both Cy3 and Cy5 by using a GenePix 4000B fluorescent scanner, and image intensity data were gridded and extracted using GenePix Pro 4.1 software. Differences in gene expression between NPF cyclin D1/NPF and CAF/NPF groups were determined using a two-sample t-test with Significance Analysis of Microarrays (SAM) software (http://www-stat.stanford.edu/_tibs/SAM/) with a False Discovery Rate (FDR) of M-bM-^IM-$ 10% considered significant (Tusher, PNAS, 2001). Similarities in gene expression between NPF cyclin D1/NPF and CAF/NPF groups were determined using a one-sample t-test in SAM with an FDR of M-bM-^IM-$ 0.1% considered significant. These results were reduced to unique genes by eliminating all but the highest scoring clones for each gene. A Pearson correlation coefficient was calculated in Excel to assess the strength of the linear relationship between NPF cyclin D1/NPF and CAF/NPF average log2 ratios.

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STIC2 selectively binds ribosome-nascent chain complexes in the cotranslational sorting of Arabidopsis thylakoid proteins

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