NAC functions as a modulator of SRP during the early steps of protein targeting to the endoplasmic reticulum

Zhang, Ying; Berndt, Uta; Gölz, Hanna; Tais, Arlette; Oellerer, Stefan; Wölfle, Tina; Fitzke, Edith; Rospert, Sabine

doi:10.1091/mbc.e12-02-0112

Cited by 62 publications

(73 citation statements)

References 66 publications

(155 reference statements)

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“…We also identified a SMYD2-mediated mono-methylation site at K1304 in RICTOR, a component of the mTORC2 kinase complex (54) as well as in the intracellular domain of GPR126, an adhesion G-protein coupled receptor that bridges type-IV collagen in the extracellular matrix to intracellular cyclic AMP signaling (55). BTF3 is a component of the evolutionary-conserved nascent polypeptide-associated complex (NAC) that prevents the inappropriate recruitment of ribosomes to the endoplasmic reticulum (56,57). The precise function of PDAP1 is unclear, but it has been reported to be highly up-regulated in the secretome of neoplastic gastric epithelial cells (58), a context in which SMYD2 activity was recently characterized (26).…”

Section: Discussionmentioning

confidence: 98%

Quantitative Profiling of the Activity of Protein Lysine Methyltransferase SMYD2 Using SILAC-Based Proteomics

Olsen

Cao

Han

et al. 2016

Molecular & Cellular Proteomics

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The significance of non-histone lysine methylation in cell biology and human disease is an emerging area of research exploration. The development of small molecule inhibitors that selectively and potently target enzymes that catalyze the addition of methyl-groups to lysine residues, such as the protein lysine mono-methyltransferase SMYD2, is an active area of drug discovery. Critical to the accurate assessment of biological function is the ability to identify target enzyme substrates and to define enzyme substrate specificity within the context of the cell. Here, using stable isotopic labeling with amino acids in cell culture (SILAC) coupled with immunoaffinity enrichment of mono-methyl-lysine (Kme1) peptides and mass spectrometry, we report a comprehensive, large-scale proteomic study of lysine mono-methylation, comprising a total of 1032 Kme1 sites in esophageal squamous cell carcinoma (ESCC) cells and 1861 Kme1 sites in ESCC cells overexpressing SMYD2. Among these Kme1 sites is a subset of 35 found to be potently down-regulated by both shRNA-mediated knockdown of SMYD2 and LLY-507, a selective small molecule inhibitor of SMYD2. In addition, we report specific protein sequence motifs enriched in Kme1 sites that are directly regulated by endogenous SMYD2 activity, revealing that SMYD2 substrate specificity is more diverse than expected. We further show direct activity of SMYD2 toward BTF3-K2, PDAP1-K126 as well as numerous sites within the repetitive units of two unique and exceptionally large proteins, AHNAK and AHNAK2. Collectively, our findings provide quantitative insights into the cellular activity and substrate recognition of SMYD2 as well as the global landscape and regulation of protein mono-methylation. Protein lysine methyltransferases (PKMTs) 1 catalyze the sequence-specific transfer of one, two, or three methyl groups to the side chains of lysine residues (1-4). In addition to the extensively studied lysine methylation on histones, PKMTs can modify non-histone proteins (3,(5)(6)(7)(8)). An increasing number of non-histone proteins have been reported as PKMT substrates, and, as a result, potential roles for the dysregulation of non-histone lysine methylation in cancer development and progression have been proposed (9). The majority of PKMT substrates have been identified based on biochemical methylation assays using recombinant enzyme and substrate, followed by cell-based assays typically requiring overexpression of enzyme and/or substrate to maximize signal detection (10 -13). However, it is difficult to discern whether these substrates are bona fide physiological substrates of endogenous PKMT activity. With the development of PKMT-targeted drugs emerging as a key area of drug discovery (14 -18), unbiased and quantitative methods enabling the comprehensive identification of histone and non-histone substrates in cells are critical to clarifying the cell-relevant substrates of these enzymes and to more accurately understand the functions of non-histone methylation.Progressing from targeted biochemical...

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Section: Discussionmentioning

confidence: 98%

Quantitative Profiling of the Activity of Protein Lysine Methyltransferase SMYD2 Using SILAC-Based Proteomics

Olsen

Cao

Han

et al. 2016

Molecular & Cellular Proteomics

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“…Plasmids encoding Dap2 (pSPUTK-Dap2) and Pgk1 (pSPUTK-Pgk1) and the FLAG-tagged versions, pSPUTK-FLAG-Dap2 and pSPUTK-FLAG-Pgk1, which contain an N-terminal DY KDDDDK FLAG peptide, were described previously (24). Genes encoding Kcv, Kesv, and Kesv-VV were amplified from plasmids pYES2-Kcv, pYES2-Kesv, and pYES2-Kesv-113VV (21) and were cloned with or without the FLAG tag into pSP65 (Promega).…”

Section: Methodsmentioning

confidence: 99%

“…Dap2-Kesv, ⌬N-Kesv, and full-length Sec22 with or without the FLAG tag were cloned into pSP65. MH272-3f a/␣ (ura3/ura3 leu2/leu2 his3/his3 trp1/trp1 ade2/ade2) was the parental wild-type strain (25), the ⌬srp54 strain was described previously (24). In the ⌬get4 ⌬get5 strain (get4:: kanMX get5::kanMX), the complete coding sequences of GET4 and GET5 were replaced by the kanMX module amplified by PCR from strain Y02420 or Y16261, respectively (Euroscarf).…”

Section: Methodsmentioning

confidence: 99%

Cotranslational Intersection between the SRP and GET Targeting Pathways to the Endoplasmic Reticulum of Saccharomyces cerevisiae

Zhang

Schäffer

Wölfle

et al. 2016

Molecular and Cellular Biology

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cTargeting of transmembrane proteins to the endoplasmic reticulum (ER) proceeds via either the signal recognition particle (SRP) or the guided entry of tail-anchored proteins (GET) pathway, consisting of Get1 to -5 and Sgt2. While SRP cotranslationally targets membrane proteins containing one or multiple transmembrane domains, the GET pathway posttranslationally targets proteins containing a single C-terminal transmembrane domain termed the tail anchor. Here, we dissect the roles of the SRP and GET pathways in the sorting of homologous, two-membrane-spanning K ؉ channel proteins termed Kcv, Kesv, and Kesv-VV. We show that Kcv is targeted to the ER cotranslationally via its N-terminal transmembrane domain, while Kesv-VV is targeted posttranslationally via its C-terminal transmembrane domain, which recruits Get4-5/Sgt2 and Get3. Unexpectedly, nascent Kcv recruited not only SRP but also the Get4-5 module of the GET pathway to ribosomes. Ribosome binding of Get4-5 was independent of Sgt2 and was strongly outcompeted by SRP. The combined data indicate a previously unrecognized cotranslational interplay between the SRP and GET pathways.

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“…31 Although the function of the NAC is poorly defined, it is thought to have roles in co-translational protein folding 32 and in protein targeting to mitochondria 33 and the endoplasmic reticulum. 34 The RAC consists of the Heat Shock Protein (Hsp) 70/40 pair Ssz1/ Zuo1, 35 which is anchored to the ribosome in close proximity to the polypeptide exit tunnel via a charged C-terminal region of Zuo1. 36,37 The RAC stimulates the ATPase activity of ribosome-associated Hsp70 Ssb chaperones to protect nascent chains from misfolding and aggregation.…”

Section: Introductionmentioning

confidence: 99%

The ribosome-associated complex antagonizes prion formation in yeast

Amor

Castanzo

Delany

et al. 2015

Prion

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ABSTRACT. The number of known fungal proteins capable of switching between alternative stable conformations is steadily increasing, suggesting that a prion-like mechanism may be broadly utilized as a means to propagate altered cellular states. To gain insight into the mechanisms by which cells regulate prion formation and toxicity we examined the role of the yeast ribosome-associated complex (RAC) in modulating both the formation of the [PSI C ] prion -an alternative conformer of Sup35 protein -and the toxicity of aggregation-prone polypeptides. The Hsp40 RAC chaperone Zuo1 anchors the RAC to ribosomes and stimulates the ATPase activity of the Hsp70 chaperone Ssb. We found that cells lacking Zuo1 are sensitive to over-expression of some aggregation-prone proteins, including the Sup35 prion domain, suggesting that co-translational protein misfolding increases in Dzuo1 strains. Consistent with this finding, Dzuo1 cells exhibit higher frequencies of spontaneous and induced prion formation. Cells expressing mutant forms of Zuo1 lacking either a C-terminal charged region required for ribosome association, or the J-domain responsible for Ssb ATPase stimulation, exhibit similarly high frequencies of prion formation. Our findings are consistent with a role for the RAC in chaperoning nascent Sup35 to regulate folding of the N-terminal prion domain as it emerges from the ribosome.

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NAC functions as a modulator of SRP during the early steps of protein targeting to the endoplasmic reticulum

Cited by 62 publications

References 66 publications

Quantitative Profiling of the Activity of Protein Lysine Methyltransferase SMYD2 Using SILAC-Based Proteomics

Quantitative Profiling of the Activity of Protein Lysine Methyltransferase SMYD2 Using SILAC-Based Proteomics

Cotranslational Intersection between the SRP and GET Targeting Pathways to the Endoplasmic Reticulum of Saccharomyces cerevisiae

The ribosome-associated complex antagonizes prion formation in yeast

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