Conditional inactivation of Aspergillus nidulans sarA(SAR1) uncovers the morphogenetic potential of regulating endoplasmic reticulum (ER) exit

• Published in: Molecular microbiology Vol. 95; no. 3; p. 491
• Main Authors: Hernández-González, Miguel, Peñalva, Miguel A, Pantazopoulou, Areti
• Format: Journal Article
• Language: English
• Published: England 01.02.2015
• Subjects: Alleles; Aspergillus nidulans - cytology; Aspergillus nidulans - genetics; Aspergillus nidulans - growth & development; Aspergillus nidulans - metabolism; Endoplasmic Reticulum - metabolism; Fungal Proteins - chemistry; Fungal Proteins - genetics; Fungal Proteins - metabolism; Golgi Apparatus - metabolism; Golgi Apparatus - ultrastructure; Hot Temperature; Microscopy; Monomeric GTP-Binding Proteins - genetics; Monomeric GTP-Binding Proteins - metabolism; Morphogenesis; Mutagens; Mutation; Phenotype; Polymerase Chain Reaction; Protein Structure, Secondary; Protein Transport; Qa-SNARE Proteins - metabolism; Temperature; Vesicular Transport Proteins - chemistry; Vesicular Transport Proteins - genetics; Vesicular Transport Proteins - metabolism
• ISSN: 1365-2958
• DOI: 10.1111/mmi.12880

In the genetic model Aspergillus 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. sarA8 results in Ser substitution for conserved P-loop Gly27. sarA5 (Trp185Cys) and sarA6 (Ser186Pro) substitutions underscore the importance of the C-terminal α-helix on SarA(Sar1) function/stability. sarA6 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 Golgi cisternae, labeled with the integral membrane syntaxins SedV(Sed5) and TlgB(Tlg2) , respectively, were rapidly dissipated by sarA6. However, whereas SedV(Sed5) was shifted toward the ER, TlgB(Tlg2) relocalized to a haze, underscoring the asymmetry of Golgi organization. This rapid Golgi dissipation that takes place after blocking anterograde COPII traffic is consistent with the cisternal maturation model. Incubation of sarA6 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.