Imaging the secretory compartments involved in the intracellular traffic of CHS-4, a class IV chitin synthase, in Neurospora crassa

Adriana M. Rico-Ramírez, Robert Roberson, Meritxell Riquelme

    Research output: Contribution to journalArticle

    1 Citation (Scopus)

    Abstract

    In Neurospora crassa hyphae the localization of all seven chitin synthases (CHSs) at the Spitzenkörper (SPK) and at developing septa has been well analyzed. Hitherto, the mechanisms of CHSs traffic and sorting from synthesis to delivery sites remain largely unexplored. In Saccharomyces cerevisiae exit of Chs3p from the endoplasmic reticulum (ER) requires chaperone Chs7p. Here, we analyzed the role of CSE-7, N. crassa Chs7p orthologue, in the biogenesis of CHS-4 (orthologue of Chs3p). In a N. crassa Δcse-7 mutant, CHS-4-GFP no longer accumulated at the SPK and septa. Instead, fluorescence was retained in hyphal subapical regions in an extensive network of elongated cisternae (NEC) referred to previously as tubular vacuoles. In a complemented strain expressing a copy of cse-7 the localization of CHS-4-GFP at the SPK and septa was restored, providing evidence that CSE-7 is necessary for the localization of CHS-4 at hyphal tips and septa. CSE-7 was revealed at delimited regions of the ER at the immediacies of nuclei, at the NEC, and remarkably also at septa and the SPK. The organization of the NEC was dependent on the cytoskeleton. SEC-63, an extensively used ER marker, and NCA-1, a SERCA-type ATPase previously localized at the nuclear envelope, were used as markers to discern the nature of the membranes containing CSE-7. Both SEC-63 and NCA-1 were found at the nuclear envelope, but also at regions of the NEC. However, at the NEC only NCA-1 co-localized extensively with CSE-7. Observations by transmission electron microscopy revealed abundant rough ER sheets and distinct electron translucent smooth flattened cisternae, which could correspond collectively to the NEC, thorough the subapical cytoplasm. This study identifies CSE-7 as the putative ER receptor for its cognate cargo, the polytopic membrane protein CHS-4, and elucidates the complexity of the ER system in filamentous fungi.

    Original languageEnglish (US)
    Pages (from-to)30-42
    Number of pages13
    JournalFungal Genetics and Biology
    Volume117
    DOIs
    StatePublished - Aug 1 2018

    Fingerprint

    Chitin Synthase
    Neurospora crassa
    Endoplasmic Reticulum
    Nuclear Envelope
    Hyphae
    Rough Endoplasmic Reticulum
    Vacuoles
    Cytoskeleton
    Transmission Electron Microscopy
    Saccharomyces cerevisiae
    Adenosine Triphosphatases
    Membrane Proteins
    Cytoplasm
    Fungi
    Fluorescence
    Electrons
    Membranes

    Keywords

    • Chitin synthase 4
    • CSE-7
    • Endoplasmic reticulum
    • Neurospora crassa
    • Spitzenkörper

    ASJC Scopus subject areas

    • Microbiology
    • Genetics

    Cite this

    Imaging the secretory compartments involved in the intracellular traffic of CHS-4, a class IV chitin synthase, in Neurospora crassa. / Rico-Ramírez, Adriana M.; Roberson, Robert; Riquelme, Meritxell.

    In: Fungal Genetics and Biology, Vol. 117, 01.08.2018, p. 30-42.

    Research output: Contribution to journalArticle

    @article{5498e397562944cc91c6d39399de359c,
    title = "Imaging the secretory compartments involved in the intracellular traffic of CHS-4, a class IV chitin synthase, in Neurospora crassa",
    abstract = "In Neurospora crassa hyphae the localization of all seven chitin synthases (CHSs) at the Spitzenk{\"o}rper (SPK) and at developing septa has been well analyzed. Hitherto, the mechanisms of CHSs traffic and sorting from synthesis to delivery sites remain largely unexplored. In Saccharomyces cerevisiae exit of Chs3p from the endoplasmic reticulum (ER) requires chaperone Chs7p. Here, we analyzed the role of CSE-7, N. crassa Chs7p orthologue, in the biogenesis of CHS-4 (orthologue of Chs3p). In a N. crassa Δcse-7 mutant, CHS-4-GFP no longer accumulated at the SPK and septa. Instead, fluorescence was retained in hyphal subapical regions in an extensive network of elongated cisternae (NEC) referred to previously as tubular vacuoles. In a complemented strain expressing a copy of cse-7 the localization of CHS-4-GFP at the SPK and septa was restored, providing evidence that CSE-7 is necessary for the localization of CHS-4 at hyphal tips and septa. CSE-7 was revealed at delimited regions of the ER at the immediacies of nuclei, at the NEC, and remarkably also at septa and the SPK. The organization of the NEC was dependent on the cytoskeleton. SEC-63, an extensively used ER marker, and NCA-1, a SERCA-type ATPase previously localized at the nuclear envelope, were used as markers to discern the nature of the membranes containing CSE-7. Both SEC-63 and NCA-1 were found at the nuclear envelope, but also at regions of the NEC. However, at the NEC only NCA-1 co-localized extensively with CSE-7. Observations by transmission electron microscopy revealed abundant rough ER sheets and distinct electron translucent smooth flattened cisternae, which could correspond collectively to the NEC, thorough the subapical cytoplasm. This study identifies CSE-7 as the putative ER receptor for its cognate cargo, the polytopic membrane protein CHS-4, and elucidates the complexity of the ER system in filamentous fungi.",
    keywords = "Chitin synthase 4, CSE-7, Endoplasmic reticulum, Neurospora crassa, Spitzenk{\"o}rper",
    author = "Rico-Ram{\'i}rez, {Adriana M.} and Robert Roberson and Meritxell Riquelme",
    year = "2018",
    month = "8",
    day = "1",
    doi = "10.1016/j.fgb.2018.03.006",
    language = "English (US)",
    volume = "117",
    pages = "30--42",
    journal = "Fungal Genetics and Biology",
    issn = "1087-1845",
    publisher = "Academic Press Inc.",

    }

    TY - JOUR

    T1 - Imaging the secretory compartments involved in the intracellular traffic of CHS-4, a class IV chitin synthase, in Neurospora crassa

    AU - Rico-Ramírez, Adriana M.

    AU - Roberson, Robert

    AU - Riquelme, Meritxell

    PY - 2018/8/1

    Y1 - 2018/8/1

    N2 - In Neurospora crassa hyphae the localization of all seven chitin synthases (CHSs) at the Spitzenkörper (SPK) and at developing septa has been well analyzed. Hitherto, the mechanisms of CHSs traffic and sorting from synthesis to delivery sites remain largely unexplored. In Saccharomyces cerevisiae exit of Chs3p from the endoplasmic reticulum (ER) requires chaperone Chs7p. Here, we analyzed the role of CSE-7, N. crassa Chs7p orthologue, in the biogenesis of CHS-4 (orthologue of Chs3p). In a N. crassa Δcse-7 mutant, CHS-4-GFP no longer accumulated at the SPK and septa. Instead, fluorescence was retained in hyphal subapical regions in an extensive network of elongated cisternae (NEC) referred to previously as tubular vacuoles. In a complemented strain expressing a copy of cse-7 the localization of CHS-4-GFP at the SPK and septa was restored, providing evidence that CSE-7 is necessary for the localization of CHS-4 at hyphal tips and septa. CSE-7 was revealed at delimited regions of the ER at the immediacies of nuclei, at the NEC, and remarkably also at septa and the SPK. The organization of the NEC was dependent on the cytoskeleton. SEC-63, an extensively used ER marker, and NCA-1, a SERCA-type ATPase previously localized at the nuclear envelope, were used as markers to discern the nature of the membranes containing CSE-7. Both SEC-63 and NCA-1 were found at the nuclear envelope, but also at regions of the NEC. However, at the NEC only NCA-1 co-localized extensively with CSE-7. Observations by transmission electron microscopy revealed abundant rough ER sheets and distinct electron translucent smooth flattened cisternae, which could correspond collectively to the NEC, thorough the subapical cytoplasm. This study identifies CSE-7 as the putative ER receptor for its cognate cargo, the polytopic membrane protein CHS-4, and elucidates the complexity of the ER system in filamentous fungi.

    AB - In Neurospora crassa hyphae the localization of all seven chitin synthases (CHSs) at the Spitzenkörper (SPK) and at developing septa has been well analyzed. Hitherto, the mechanisms of CHSs traffic and sorting from synthesis to delivery sites remain largely unexplored. In Saccharomyces cerevisiae exit of Chs3p from the endoplasmic reticulum (ER) requires chaperone Chs7p. Here, we analyzed the role of CSE-7, N. crassa Chs7p orthologue, in the biogenesis of CHS-4 (orthologue of Chs3p). In a N. crassa Δcse-7 mutant, CHS-4-GFP no longer accumulated at the SPK and septa. Instead, fluorescence was retained in hyphal subapical regions in an extensive network of elongated cisternae (NEC) referred to previously as tubular vacuoles. In a complemented strain expressing a copy of cse-7 the localization of CHS-4-GFP at the SPK and septa was restored, providing evidence that CSE-7 is necessary for the localization of CHS-4 at hyphal tips and septa. CSE-7 was revealed at delimited regions of the ER at the immediacies of nuclei, at the NEC, and remarkably also at septa and the SPK. The organization of the NEC was dependent on the cytoskeleton. SEC-63, an extensively used ER marker, and NCA-1, a SERCA-type ATPase previously localized at the nuclear envelope, were used as markers to discern the nature of the membranes containing CSE-7. Both SEC-63 and NCA-1 were found at the nuclear envelope, but also at regions of the NEC. However, at the NEC only NCA-1 co-localized extensively with CSE-7. Observations by transmission electron microscopy revealed abundant rough ER sheets and distinct electron translucent smooth flattened cisternae, which could correspond collectively to the NEC, thorough the subapical cytoplasm. This study identifies CSE-7 as the putative ER receptor for its cognate cargo, the polytopic membrane protein CHS-4, and elucidates the complexity of the ER system in filamentous fungi.

    KW - Chitin synthase 4

    KW - CSE-7

    KW - Endoplasmic reticulum

    KW - Neurospora crassa

    KW - Spitzenkörper

    UR - http://www.scopus.com/inward/record.url?scp=85048732811&partnerID=8YFLogxK

    UR - http://www.scopus.com/inward/citedby.url?scp=85048732811&partnerID=8YFLogxK

    U2 - 10.1016/j.fgb.2018.03.006

    DO - 10.1016/j.fgb.2018.03.006

    M3 - Article

    VL - 117

    SP - 30

    EP - 42

    JO - Fungal Genetics and Biology

    JF - Fungal Genetics and Biology

    SN - 1087-1845

    ER -