Zinc regulates ERp44-dependent protein quality control in the early secretory pathway

Watanabe, Satoshi; Amagai, Yuta; Sannino, Sara; Tempio, Tiziana; Anelli, Tiziana; Harayama, Manami; Masui, Shoji; Sorrentino, Ilaria; Yamada, Momo; Sitia, Roberto; Inaba, Kenji

doi:10.1038/s41467-019-08429-1

Cited by 55 publications

(60 citation statements)

References 81 publications

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“…In contrast, wild type Gas1 bypasses endosomes on its way to the plasma membrane (Figure 7). These data are in agreement with previous reports about the relevance of the Golgi in post-ER quality control and in directing substrates to the vacuole for degradation (Arvan et al, 2002;Vashist et al, 2001;Wang and Ng, 2010;Watanabe et al, 2019;Dobzinski et al, 2015). It is unclear at present which signal is recognized inside the Golgi and what mechanism diverts Gas1* to endosomes.…”

Section: Discussionsupporting

confidence: 93%

A post-ER degradation pathway that relies on protease-dependent internalization from the vacuolar membrane

Lemus

Matić

Goder

2019

Preprint

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2 Abbreviations: GPI anchor: glycosylphophatidylinositol anchor; ESCRT: endosomal sorting complexes required for transport; MVBs: multi vesicular bodies; ILVs: intraluminal vesicles; ERAD: endoplasmic reticulum-associated protein degradation; TMD: transmembrane domain; PVC: prevacuolar compartment; SNARE: soluble NSF attachment protein receptor; Cvt vesicle: cytoplasm to vacuole targeting vesicle Keywords: Protein degradation, secretory pathway, vacuole, vacuolar protease, membrane invagination Highlights: • ER-exited misfolded GPI-anchored proteins are routed to the vacuole via endosomes but do not internalize into intraluminal vesicles • Internalization occurs directly from the vacuolar membrane into intravacuolar mobile structures • Internalization from the vacuolar membrane depends on the proteolytic activity of the vacuolar protease Pep4 3 Summary Newly synthesized proteins of the secretory pathway are quality-controlled inside the endoplasmic reticulum (ER) and, if not properly folded, are retained. An exception are glycosylphosphatidylinositol-anchored proteins (GPI-APs) which can leave the ER even when misfolded and are routed to the vacuole/lysosome for degradation by largely unknown mechanisms linked to post-ER quality control. Using yeast as model organism,we show that Gas1*, an ER-exported misfolded GPI-AP, is diverted from the secretory pathway to endosomes for transport to the vacuole. However, Gas1* is not sorted into endosomal intraluminal vesicles but internalizes directly from the vacuolar membrane.There, the vacuolar protease Pep4, but not any other known vacuolar protease, is required for Gas1* internalization. Our data reveal novel and unexpected mechanisms for invaginations from the vacuolar membrane.

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Section: Discussionsupporting

confidence: 93%

A post-ER degradation pathway that relies on protease-dependent internalization from the vacuolar membrane

Lemus

Matić

Goder

2019

Preprint

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“…Considering the differences between PLAP and secPLAP, the addition of the GPI anchor might facilitate the protein degradation, and addressing this question could provide information regarding the manner in which ZNT5-ZNT6 heterodimers and ZNT7 homodimers contribute to homeostatic maintenance in the early secretory pathway (48,51). However, other possibilities also exist, such as homeostatic maintenance being achieved through the regulation of chaperone-protein functions by zinc that is mediated by both complexes (60,61). Disruption of Znt and/or Zip genes in DT40 cells and ZNT genes in HAP1 cells increased the mRNA expression of other Znt and/or Zip genes in DT40 cells and ZNT genes in HAP1 cells (Figs.…”

Section: Discussionmentioning

confidence: 99%

Detailed analyses of the crucial functions of Zn transporter proteins in alkaline phosphatase activation

Suzuki

Ogawa

Takeda

et al. 2020

Journal of Biological Chemistry

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Numerous zinc ectoenzymes are metalated by zinc and activated in the compartments of the early secretory pathway before reaching their destination. Zn transporter (ZNT) proteins located in these compartments are essential for ectoenzyme activation. We have previously reported that ZNT proteins, specifically ZNT5–ZNT6 heterodimers and ZNT7 homodimers, play critical roles in the activation of zinc ectoenzymes, such as alkaline phosphatases (ALPs), by mobilizing cytosolic zinc into these compartments. However, this process remains incompletely understood. Here, using genetically-engineered chicken DT40 cells, we first determined that Zrt/Irt-like protein (ZIP) transporters that are localized to the compartments of the early secretory pathway play only a minor role in the ALP activation process. These transporters included ZIP7, ZIP9, and ZIP13, performing pivotal functions in maintaining cellular homeostasis by effluxing zinc out of the compartments. Next, using purified ALP proteins, we showed that zinc metalation on ALP produced in DT40 cells lacking ZNT5–ZNT6 heterodimers and ZNT7 homodimers is impaired. Finally, by genetically disrupting both ZNT5 and ZNT7 in human HAP1 cells, we directly demonstrated that the tissue-nonspecific ALP-activating functions of both ZNT complexes are conserved in human cells. Furthermore, using mutant HAP1 cells, we uncovered a previously-unrecognized and unique spatial regulation of ZNT5–ZNT6 heterodimer formation, wherein ZNT5 recruits ZNT6 to the Golgi apparatus to form the heterodimeric complex. These findings fill in major gaps in our understanding of the molecular mechanisms underlying zinc ectoenzyme activation in the compartments of the early secretory pathway.

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“…Anti-Flag antibody (M2) is from Sigma 69 . Anti-MEK5 antibody (AB3184) is from EMD Millipore 22 , anti-lamin A/C (636) 70 , ERK5 (C-7) 26 and myc (9E10) 71 antibodies are from Santa Cruz and anti-MEK1/2 72 , phospho-T359 RSK 73 and total RSK 74 from Cell Signaling Technology. Secondary antibodies were from BioRad 40,41 .…”

Section: Methodsmentioning

confidence: 99%

Paradoxical activation of the protein kinase-transcription factor ERK5 by ERK5 kinase inhibitors

et al. 2020

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The dual protein kinase-transcription factor, ERK5, is an emerging drug target in cancer and inflammation, and small-molecule ERK5 kinase inhibitors have been developed. However, selective ERK5 kinase inhibitors fail to recapitulate ERK5 genetic ablation phenotypes, suggesting kinase-independent functions for ERK5. Here we show that ERK5 kinase inhibitors cause paradoxical activation of ERK5 transcriptional activity mediated through its unique C-terminal transcriptional activation domain (TAD). Using the ERK5 kinase inhibitor, Compound 26 (ERK5-IN-1), as a paradigm, we have developed kinase-active, drug-resistant mutants of ERK5. With these mutants, we show that induction of ERK5 transcriptional activity requires direct binding of the inhibitor to the kinase domain. This in turn promotes conformational changes in the kinase domain that result in nuclear translocation of ERK5 and stimulation of gene transcription. This shows that both the ERK5 kinase and TAD must be considered when assessing the role of ERK5 and the effectiveness of anti-ERK5 therapeutics. 1 1234567890():,;E xtracellular signal-regulated kinase 5 (ERK5, also known as Big MAP Kinase or BMK1) 1,2 is a member of the mitogenactivated protein kinase (MAPK) family, which also includes ERK1/2 3 , the JNKs 4 and the p38 kinases 5 . Like ERK1/2, ERK5 is the effector kinase of a three-tiered MAPK pathway, comprising MEKK2 and MEKK3 (the MKKKs), MEK5 (MKK) and finally ERK5 (MAPK). ERK5 is encoded by the MAPK7 gene and includes an N-terminal kinase domain that shares 50% identity with ERK2 1,2 . However, it also contains a large, unique C-terminal extension that includes a nuclear localisation signal (NLS) and a transcriptional activation domain (TAD) (Fig. 1a) 6 . The ERK5 pathway is activated by mitogens 7 , agonists of the Tolllike receptor-2 8 and cellular stresses 9 . Upon cellular stimulation, activated MEK5 phosphorylates the TEY motif in the ERK5 activation-loop, leading to activation of its kinase domain 10 . The ERK5 C-terminus also becomes auto-phosphorylated and promotes ERK5 translocation from the cytosol to the nucleus 11,12 , where ERK5 has been shown to interact with MEF2 transcription factors such as MEF2D 7,13,14 . The C-terminus can also be regulated by other protein kinases, including ERK1/2 15 and CDK1 16,17 , which phosphorylate C-terminal residues independently of ERK5 kinase activity. Thus, the C-terminus mediates NATURE COMMUNICATIONS | https://doi.

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Zinc regulates ERp44-dependent protein quality control in the early secretory pathway

Cited by 55 publications

References 81 publications

A post-ER degradation pathway that relies on protease-dependent internalization from the vacuolar membrane

A post-ER degradation pathway that relies on protease-dependent internalization from the vacuolar membrane

Detailed analyses of the crucial functions of Zn transporter proteins in alkaline phosphatase activation

Paradoxical activation of the protein kinase-transcription factor ERK5 by ERK5 kinase inhibitors

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