Cell viability and secretion of active proteins in Schizosaccharomyces pombe do not require the chaperone function of calnexin

Alexandre MARÉCHAL; Pierre-Luc TANGUAY; Mario CALLEJO; Renée GUÉRIN; Guy BOILEAU; Luis A. ROKEACH

doi:10.1042/bj20031546

Cell viability and secretion of active proteins in Schizosaccharomyces pombe do not require the chaperone function of calnexin

Biochemical Journal ◽

10.1042/bj20031546 ◽

2004 ◽

Vol 380 (2) ◽

pp. 441-448 ◽

Cited By ~ 14

Author(s):

Alexandre MARÉCHAL ◽

Pierre-Luc TANGUAY ◽

Mario CALLEJO ◽

Renée GUÉRIN ◽

Guy BOILEAU ◽

...

Keyword(s):

Quality Control ◽

Schizosaccharomyces Pombe ◽

Secretory Pathway ◽

Cellular Processes ◽

Rate Limiting Step ◽

Secretion Efficiency ◽

Model Protein ◽

Endogenous Proteins ◽

Quality Control Mechanism

Folding of newly synthesized proteins within the ER (endoplasmic reticulum) is a rate-limiting step in protein secretion. Thus ER molecular chaperones and foldases have a major impact in determining the rate and yield of these crucial cellular processes. Calnexin is a key ER chaperone implicated in the folding, retention and targeting for degradation of proteins that go through the secretory pathway. Calnexin molecules contain a highly conserved central domain (hcd) that has been proposed to be involved in the interaction with folding substrates and other chaperones. To gain a better understanding of the roles played by calnexin in the secretory pathway, we examined the efficiency of fission yeast (Schizosaccharomyces pombe) strains expressing calnexin mutants to secrete different model proteins. Remarkably, calnexin hcd-deletion mutants, although devoid of detectable chaperone activity in vitro, confer viability and cause a considerable increase in the secretion of heterologous cellulase. Surprisingly the quality-control efficiency, measured as the activity/amount ratio of secreted model protein, was not severely reduced in these calnexin hcd-deletion mutant strains. Our results indicate that the essential function of calnexin does not reside in its role in the folding or in the retention of misfolded proteins. These observations suggest the existence of a highly stringent quality control mechanism in the ER of S. pombe that might reduce the secretion efficiency of endogenous proteins.

Download Full-text

Quality Control of a Transcriptional Regulator by SUMO-Targeted Degradation

Molecular and Cellular Biology ◽

10.1128/mcb.01470-08 ◽

2009 ◽

Vol 29 (7) ◽

pp. 1694-1706 ◽

Cited By ~ 55

Author(s):

Zheng Wang ◽

Gregory Prelich

Keyword(s):

Quality Control ◽

Protein Quality ◽

Temperature Sensitive ◽

Wild Type ◽

Physiological Importance ◽

Sensitive Allele ◽

Quality Control Mechanism ◽

Mutant Phenotypes

ABSTRACT Slx5 and Slx8 are heterodimeric RING domain-containing proteins that possess SUMO-targeted ubiquitin ligase (STUbL) activity in vitro. Slx5-Slx8 and its orthologs are proposed to target SUMO conjugates for ubiquitin-mediated proteolysis, but the only in vivo substrate identified to date is mammalian PML, and the physiological importance of SUMO-targeted ubiquitylation remains largely unknown. We previously identified mutations in SLX5 and SLX8 by selecting for suppressors of a temperature-sensitive allele of MOT1, which encodes a regulator of TATA-binding protein. Here, we demonstrate that Mot1 is SUMOylated in vivo and that disrupting the Slx5-Slx8 pathway by mutation of the target lysines in Mot1, by deletion of SLX5 or the ubiquitin E2 UBC4, or by inhibition of the proteosome suppresses mot1-301 mutant phenotypes and increases the stability of the Mot1-301 protein. The Mot1-301 mutant protein is targeted for proteolysis by SUMOylation to a much greater extent than wild-type Mot1, suggesting a quality control mechanism. In support of this idea, growth of Saccharomyces cerevisiae in the presence of the arginine analog canavanine results in increased SUMOylation and Slx5-Slx8-mediated degradation of wild-type Mot1. These results therefore demonstrate that Mot1 is an in vivo STUbL target in yeast and suggest a role for SUMO-targeted degradation in protein quality control.

Download Full-text

A putative, ubiquitin-dependent mechanism for the recognition and elimination of defective spermatozoa in the mammalian epididymis

Journal of Cell Science ◽

10.1242/jcs.114.9.1665 ◽

2001 ◽

Vol 114 (9) ◽

pp. 1665-1675 ◽

Cited By ~ 4

Author(s):

P. Sutovsky ◽

R. Moreno ◽

J. Ramalho-Santos ◽

T. Dominko ◽

W.E. Thompson ◽

...

Keyword(s):

Quality Control ◽

Control Mechanism ◽

Sperm Quality ◽

Normal Structure ◽

Structure And Function ◽

Quality Control Mechanism ◽

And Function ◽

And Storage ◽

Dependent Mechanism

The normal structure and function of sperm are prerequisites for successful fertilization and embryonic development, but little is known about how defective sperm are eliminated during mammalian spermatogenesis. Here, we describe a ubiquitin-dependent, sperm quality control mechanism that resides in the mammalian epididymis, the site of sperm maturation and storage. We used immunofluorescence, electron microscopy, western blotting and pulse-chase experiments to show that ubiquitin is secreted by the epididymal epithelium and binds to the surface of defective sperm. Most of the ubiquitinated sperm are subsequently phagocytosed by the epididymal epithelial cells. A portion of defective sperm escapes phagocytosis and can be found in the ejaculate. Cultured epididymal cells maintain their ability to produce ubiquitin and phagocytose the defective sperm, as well as the ubiquitin-coated microspheres, in vitro. The surprising phenomenon of cell-surface ubiquitination in defective sperm provides a possible mechanism for sperm quality control in mammals and a new marker of semen abnormalities in men and animals.

Download Full-text

Presynaptic Vesicle Protein SEPTIN5 Regulates the Degradation of APP C-Terminal Fragments and the Levels of Aβ

Cells ◽

10.3390/cells9112482 ◽

2020 ◽

Vol 9 (11) ◽

pp. 2482

Author(s):

Mikael Marttinen ◽

Catarina B. Ferreira ◽

Kaisa M. A. Paldanius ◽

Mari Takalo ◽

Teemu Natunen ◽

...

Keyword(s):

Secretory Pathway ◽

Neuronal Cells ◽

Amyloid Β ◽

In Vivo Studies ◽

App Processing ◽

Cellular Processes ◽

Presynaptic Vesicle ◽

Tau Aggregation

Alzheimer’s disease (AD) is a neurodegenerative disease characterized by aberrant amyloid-β (Aβ) and hyperphosphorylated tau aggregation. We have previously investigated the involvement of SEPTIN family members in AD-related cellular processes and discovered a role for SEPTIN8 in the sorting and accumulation of β-secretase. Here, we elucidated the potential role of SEPTIN5, an interaction partner of SEPTIN8, in the cellular processes relevant for AD, including amyloid precursor protein (APP) processing and the generation of Aβ. The in vitro and in vivo studies both revealed that the downregulation of SEPTIN5 reduced the levels of APP C-terminal fragments (APP CTFs) and Aβ in neuronal cells and in the cortex of Septin5 knockout mice. Mechanistic elucidation revealed that the downregulation of SEPTIN5 increased the degradation of APP CTFs, without affecting the secretory pathway-related trafficking or the endocytosis of APP. Furthermore, we found that the APP CTFs were degraded, to a large extent, via the autophagosomal pathway and that the downregulation of SEPTIN5 enhanced autophagosomal activity in neuronal cells as indicated by altered levels of key autophagosomal markers. Collectively, our data suggest that the downregulation of SEPTIN5 increases the autophagy-mediated degradation of APP CTFs, leading to reduced levels of Aβ in neuronal cells.

Download Full-text

Distinct retrieval and retention mechanisms are required for the quality control of endoplasmic reticulum protein folding

The Journal of Cell Biology ◽

10.1083/jcb.200106123 ◽

2001 ◽

Vol 155 (3) ◽

pp. 355-368 ◽

Cited By ~ 176

Author(s):

Shilpa Vashist ◽

Woong Kim ◽

William J. Belden ◽

Eric D. Spear ◽

Charles Barlowe ◽

...

Keyword(s):

Quality Control ◽

Endoplasmic Reticulum ◽

Secretory Pathway ◽

Protein Translocation ◽

Retrograde Transport ◽

26S Proteasome ◽

Misfolded Proteins ◽

Mutant Proteins ◽

Endoplasmic Reticulum Protein ◽

Quality Control Mechanism

Proteins destined for the secretory pathway must first fold and assemble in the lumen of endoplasmic reticulum (ER). The pathway maintains a quality control mechanism to assure that aberrantly processed proteins are not delivered to their sites of function. As part of this mechanism, misfolded proteins are returned to the cytosol via the ER protein translocation pore where they are ubiquitinated and degraded by the 26S proteasome. Previously, little was known regarding the recognition and targeting of proteins before degradation. By tracking the fate of several mutant proteins subject to quality control, we demonstrate the existence of two distinct sorting mechanisms. In the ER, substrates are either sorted for retention in the ER or are transported to the Golgi apparatus via COPII–coated vesicles. Proteins transported to the Golgi are retrieved to the ER via the retrograde transport system. Ultimately, both retained and retrieved proteins converge at a common machinery at the ER for degradation. Furthermore, we report the identification of a gene playing a novel role specific to the retrieval pathway. The gene, BST1, is required for the transport of misfolded proteins to the Golgi, although dispensable for the transport of many normal cargo proteins.

Download Full-text

Localization of an alpha 1,2 galactosyltransferase activity to the Golgi apparatus of Schizosaccharomyces pombe.

Molecular Biology of the Cell ◽

10.1091/mbc.5.5.519 ◽

1994 ◽

Vol 5 (5) ◽

pp. 519-528 ◽

Cited By ~ 39

Author(s):

T G Chappell ◽

M A Hajibagheri ◽

K Ayscough ◽

M Pierce ◽

G Warren

Keyword(s):

Golgi Apparatus ◽

Schizosaccharomyces Pombe ◽

Intracellular Transport ◽

Secretory Pathway ◽

Lectin Binding ◽

Yeast Cells ◽

Homologous Gene ◽

Carbohydrate Structure ◽

Electron Microscopic

We have cloned a gene encoding an alpha 1,2 galactosyltransferase activity from Schizosaccharomyces pombe. The open reading frame of the gene (gma12 for galactomannan, alpha 1,2), combined with the previous protein purification (Chappell and Warren, 1989), predicts an O-linked glycoprotein with type II transmembrane topology. By homologous gene disruption, we have demonstrated that the gma12 gene product (gma12p) is nonessential. The deletion strain (gma12-D10::ura4) has a significantly reduced level of galactosyltransferase activity relative to the parental strain, but both in situ lectin binding and in vitro biochemical assays demonstrate the presence of further galactosyltransferase activity in addition to gma12p. Although gma12p is not the only galactosyltransferase in S. pombe, it produces a unique carbohydrate structure on the surface of the yeast cells. We have generated a polyclonal antiserum against this carbohydrate epitope and shown that gma12p is capable of synthesizing the epitope both in vitro and in vivo. Electron microscopic localization of the gma12+ specific epitope in gma12+ cells revealed that gma12p synthesizes the carbohydrate structure in the Golgi apparatus, and subsequent intracellular transport distributes the epitope to later stages of the secretory pathway. The immunolocalization studies confirm the presence of one or more galactosyltransferase activities in the Golgi apparatus in fission yeast.

Download Full-text

Endoplasmic reticulum acetyltransferases Atase1 and Atase2 differentially regulate reticulophagy, macroautophagy and cellular acetyl-CoA metabolism

Communications Biology ◽

10.1038/s42003-021-01992-8 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Michael J. Rigby ◽

Alexis J. Lawton ◽

Gulpreet Kaur ◽

Varuna C. Banduseela ◽

William E. Kamm ◽

...

Keyword(s):

Quality Control ◽

Secretory Pathway ◽

Control Mechanism ◽

Cellular Biology ◽

Lysine Acetylation ◽

Type Ii ◽

Acetyl Coa ◽

Toxic Protein ◽

Quality Control Mechanism

AbstractNε-lysine acetylation in the ER lumen is a recently discovered quality control mechanism that ensures proteostasis within the secretory pathway. The acetyltransferase reaction is carried out by two type-II membrane proteins, ATase1/NAT8B and ATase2/NAT8. Prior studies have shown that reducing ER acetylation can induce reticulophagy, increase ER turnover, and alleviate proteotoxic states. Here, we report the generation of Atase1−/− and Atase2−/− mice and show that these two ER-based acetyltransferases play different roles in the regulation of reticulophagy and macroautophagy. Importantly, knockout of Atase1 alone results in activation of reticulophagy and rescue of the proteotoxic state associated with Alzheimer’s disease. Furthermore, loss of Atase1 or Atase2 results in widespread adaptive changes in the cell acetylome and acetyl-CoA metabolism. Overall, our study supports a divergent role of Atase1 and Atase2 in cellular biology, emphasizing ATase1 as a valid translational target for diseases characterized by toxic protein aggregation in the secretory pathway.

Download Full-text