The specific subcellular localization of two isoforms of cytochrome b5 suggests novel targeting pathways.

D’Arrigo, Antonello; Manera, Ernesto; Longhi, Renato; Borgese, Nica

doi:10.1016/s0021-9258(18)53844-8

Cited by 70 publications

(7 citation statements)

References 39 publications

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“…Knockdown of SEC61A1 also produced a substantial growth defect. CYB5B is a second TA protein known to localize to both the ER and mitochondria ( D’Arrigo et al, 1993 ). It was seen to decrease HiLITR activation in the TA and SA screens and increase HiLITR activation in the ER screen ( C–E ).…”

Section: Resultsmentioning

confidence: 99%

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An engineered transcriptional reporter of protein localization identifies regulators of mitochondrial and ER membrane protein trafficking in high-throughput CRISPRi screens

Coukos

Yao

Sanchez

et al. 2021

eLife

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The trafficking of specific protein cohorts to correct subcellular locations at correct times is essential for every signaling and regulatory process in biology. Gene perturbation screens could provide a powerful approach to probe the molecular mechanisms of protein trafficking, but only if protein localization or mislocalization can be tied to a simple and robust phenotype for cell selection, such as cell proliferation or fluorescence-activated cell sorting (FACS). To empower the study of protein trafficking processes with gene perturbation, we developed a genetically-encoded molecular tool named HiLITR. HiLITR converts protein colocalization into proteolytic release of a membrane-anchored transcription factor, which drives the expression of a chosen reporter gene. Using HiLITR in combination with FACS-based CRISPRi screening in human cell lines, we identified genes that influence the trafficking of mitochondrial and ER tail-anchored proteins. We show that loss of the SUMO E1 component SAE1 results in mislocalization and destabilization of many mitochondrial tail-anchored proteins. We also demonstrate a distinct regulatory role for EMC10 in the ER membrane complex, opposing the transmembrane-domain insertion activity of the complex. Through transcriptional integration of complex cellular functions, HiLITR expands the scope of biological processes that can be studied by genetic perturbation screening technologies.

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Section: Resultsmentioning

confidence: 99%

“…CYB5B is a second tail-anchored protein known to localize to both the ER and mitochondria (D'Arrigo et al, 1993). It was seen to decrease HiLITR activation in the TA and SA screens and increase HiLITR activation in the ER screen (Figure 3 figure supplement 2C-E).…”

Section: Figurefigure Supplement 1 Re-testing Of Trc/get Pathway Genes With Hilitrmentioning

confidence: 90%

An engineered transcriptional reporter of protein localization identifies regulators of mitochondrial and ER membrane protein trafficking in high-throughput CRISPRi screens

Coukos

Yao

Sanchez

et al. 2021

eLife

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“…Liposomes were reconstituted from control membranes following protein removal (Figure 3A: compare lanes 1 and 2). Unlike Cytb5 that has been reported to efficiently bind to lipid (33), only 5% of Nyv1p bound to liposomes (Figure 35 S-labeled prepro-alpha factor was incubated with yeast microsomes (lanes 1-5), as described under Experimental Procedures, and membrane-associated material was sedimented by ultracentrifugation. Samples representing equal amounts of the total (lane 1), soluble (lane 2), membranebound (lane 3), carbonate-soluble (lanes 4), and carbonate-resistant fractions (lane 5) were analyzed by SDS-PAGE and phosphorimaging.…”

Section: Resultsmentioning

confidence: 99%

Tail-Anchored Protein Insertion into Yeast ER Requires a Novel Posttranslational Mechanism Which Is Independent of the SEC Machinery

2002

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Tail-anchored or C-terminally-anchored proteins play many essential roles in eukaryotic cells. However, targeting and insertion of this class of membrane protein has remained elusive. In this study, we reconstitute insertion of tail-anchored proteins into microsomes derived from Saccharomyces cerevisiae. Using this approach, we are able to genetically manipulate the composition of the microsomes in order to address the question of which components of the endoplasmic reticulum (ER) are required for this process. We show that tail-anchored protein insertion is not dependent on the classical SEC translocation machinery but rather occurs via an ATP-dependent pathway involving at least one novel membrane protein factor. We further demonstrate that the specificity of this pathway is conserved between yeast and mammals.

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“…Therefore, the carboxylterminal hydrophobic sequence was termed an "insertion" sequence through which spontaneous integration of cytochrome b5 into any exposed membranes could occur not only in vitro, but also in vivo (5). However, recent studies have shown that cytochrome b5 is restricted in the ER in vivo (11), and that the last 10 amino acids of this protein adjacent to the hydrophobic domain are important for its targeting to the ER (23).…”

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confidence: 99%

Microsomal aldehyde dehydrogenase is localized to the endoplasmic reticulum via its carboxyl-terminal 35 amino acids.

Masaki

Yamamoto

Tashiro

1994

The Journal of Cell Biology

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Abstract. Rat microsomal aldehyde dehydrogenase (msALDH) has no amino-terminal signal sequence, but instead it has a characteristic hydrophobic domain at the carboxyl terminus (Miyauehi, K., R. Masaki, S. Taketani, A. Yamamoto, A. Akayama, and Y. Tashiro. 1991. J. Biol. Chem. 266:19536-19542). This membrane-bound enzyme is a useful model protein for studying posttranslational localization to its final destination. When expressed from eDNA in COS-1 cells, wild-type msALDH is localized exclusively in the well-developed ER. The removal of the hydrophobic domain results in the cytosolic localization of truncated proteins, thus suggesting that the portion is responsible for membrane anchoring. The last 35 amino acids of msALDH, including the hydrophobic domain, are sufficient for targeting of E. coli ~5-galactosidase to the ER membrane. Further studies using chloramphenicol acetyltransferase fusion proteins suggest that two hydrophilie sequences on either side of the hydrophobic domain play an important role in ER targeting.great deal of attention has been paid to resolve the mechanism for sorting and targeting of newly synthesized proteins to their final destinations, and this is one of the fundamental problemsin cell biology. Newly synthesized proteins are destined to follow two distinct routes, depending on the presence or absence of a signal sequence at their amino termini (37). The signal recognition particle (SRP) t in the cytosol recognizes and binds to a signal sequence of nascent peptides. The resulting SRP-ribosome-nascent peptides complexes are targeted to the ER membrane through their interactions with the docking protein complex (35,36). After translocation across the ER membrane, secretory and plasma membrane proteins follow a common pathway from the ER, through the Golgi complex, to the cell surface by default with bulk flow of lipids (33). Resident proteins in the central vacuolar system are localized and retained in their final destinations with the aid of either specific targeting (18) or retention signals (25)(26)(27)44).On the other hand, proteins without a signal sequence at their amino termini are synthesized on free polysomes and

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The specific subcellular localization of two isoforms of cytochrome b5 suggests novel targeting pathways.

Cited by 70 publications

References 39 publications

An engineered transcriptional reporter of protein localization identifies regulators of mitochondrial and ER membrane protein trafficking in high-throughput CRISPRi screens

An engineered transcriptional reporter of protein localization identifies regulators of mitochondrial and ER membrane protein trafficking in high-throughput CRISPRi screens

Tail-Anchored Protein Insertion into Yeast ER Requires a Novel Posttranslational Mechanism Which Is Independent of the SEC Machinery

Microsomal aldehyde dehydrogenase is localized to the endoplasmic reticulum via its carboxyl-terminal 35 amino acids.

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