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Conditional inactivation of A spergillus nidulans sarA SAR1 uncovers the morphogenetic potential of regulating endoplasmic reticulum (ER) exit
Author(s) -
HernándezGonzález Miguel,
Peñalva Miguel A.,
Pantazopoulou Areti
Publication year - 2015
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/mmi.12880
Subject(s) - endoplasmic reticulum , biology , copii , microbiology and biotechnology , wild type , er retention , vesicular transport proteins , mutant , secretory pathway , golgi apparatus , gene , genetics , cytoplasm , vacuole , vacuolar protein sorting
Summary In the genetic model A spergillus nidulans , hyphal growth is exquisitely dependent on exocytic traffic. Following mutagenic PCR and gene replacement, we characterized thermosensitive mutations in sarA SAR1 encoding a key regulator of endoplasmic reticulum ( ER ) exit. Six sarA ts alleles permitting relatively normal growth at 30°C prevented it at 42°C. This growth phenotype correlated with markedly reduced SarA levels at high temperature, suggesting that these alleles cause temperature‐dependent SarA misfolding. sarA 8 results in Ser substitution for conserved P ‐loop G ly27. sarA 5 ( T rp185 C ys) and sarA 6 ( S er186 P ro) substitutions underscore the importance of the C ‐terminal α‐helix on SarA Sar1 function/stability. sarA 6 markedly diminishing growth at 37°C was useful for microscopy experiments in which ER exit was impaired by shifting the incubation temperature. Early and late G olgi cisternae, labeled with the integral membrane syntaxins SedV Sed5 and TlgB Tlg2 , respectively, were rapidly dissipated by sarA 6. However, whereas SedV Sed5 was shifted toward the ER , TlgB Tlg2 relocalized to a haze, underscoring the asymmetry of G olgi organization. This rapid G olgi dissipation that takes place after blocking anterograde COPII traffic is consistent with the cisternal maturation model. Incubation of sarA 6 cells at 37°C led to the formation of apical balloons resembling specialized fungal structures. The formation of these balloons highlights the morphogenetic consequences of impairing ER exit.