A Fungal Sarcolemmal Membrane-Associated Protein (SLMAP) Homolog Plays a Fundamental Role in Development and Localizes to the Nuclear Envelope, Endoplasmic Reticulum, and Mitochondria

Abstract

bSarcolemmal membrane-associated protein (SLMAP) is a tail-anchored protein involved in fundamental cellular processes, such as myoblast fusion, cell cycle progression, and chromosomal inheritance. Further, SLMAP misexpression is associated with endothelial dysfunctions in diabetes and cancer. SLMAP is part of the conserved striatin-interacting phosphatase and kinase (STRIPAK) complex required for specific signaling pathways in yeasts, filamentous fungi, insects, and mammals. In filamentous fungi, STRIPAK was initially discovered in Sordaria macrospora, a model system for fungal differentiation. Here, we functionally characterize the STRIPAK subunit PRO45, a homolog of human SLMAP. We show that PRO45 is required for sexual propagation and cell-to-cell fusion and that its forkhead-associated (FHA) domain is essential for these processes. Protein-protein interaction studies revealed that PRO45 binds to STRIPAK subunits PRO11 and SmMOB3, which are also required for sexual propagation. Superresolution structured-illumination microscopy (SIM) further established that PRO45 localizes to the nuclear envelope, endoplasmic reticulum, and mitochondria. SIM also showed that localization to the nuclear envelope requires STRIPAK subunits PRO11 and PRO22, whereas for mitochondria it does not. Taken together, our study provides important insights into fundamental roles of the fungal SLMAP homolog PRO45 and suggests STRIPAK-related and STRIPAK-unrelated functions. Membrane recruitment of protein complexes, cell signaling modules, and enzymes is a critical step for many cellular functions. The family of tail-anchored proteins is recognized for anchoring proteins and vesicles to specific membranes, such as the endoplasmic reticulum (ER) and the outer mitochondrial membrane (1), and tail-anchored proteins are characterized by a C-terminal single transmembrane domain, which is posttranslationally inserted into membranes (2, 3).Sarcolemmal membrane-associated protein (SLMAP) is a tailanchored protein first identified in myocardiac cells (4). In mammals, this protein is known to be involved in myoblast fusion during embryonic development, excitation-contraction coupling in cardiac myocytes, and cell cycle progression (5-8). Furthermore, SLMAP was identified to be a disease gene for Brugada syndrome, a cardiac channelopathy (9). The functional diversity of SLMAP is dependent on alternative splicing, leading to at least four different isoforms of the protein (4, 6, 7, 10). Importantly, gene expression analyses have implicated SLMAP misexpression with endothelial dysfunctions in diabetes, chromosomal aberrations, and cancer (11-14), and currently, SLMAP is the target of lectin-based treatment of drug-resistant cancer cells (15).SLMAP is conserved from yeasts to humans, and characterized fungal SLMAP homologs include Neurospora crassa HAM-4 (hyphal anastomosis 4), Saccharomyces cerevisiae Far9p (factor arrest 9p) and Far10p, as well as Schizosaccharomyces pombe Csc1p (component of SIP complex 1p) (16-18). HAM-4 is essential for vegetativ...

Signal exchange and integration during self-fusion in filamentous fungi

“…The essential role of these factors in anastomosis formation was first described for S. macrospora and N. crassa and has also been confirmed in the plant pathogenic species Colletotrichum graminicola [54,[57][58][59][60][61]. STRIPAK functions are complex, especially because its core can associate and interact with various other proteins, and individual factors of the complex can possess STRIPAK-independent roles.…”

“…Consistent with their various tasks, STRIPAK factors localize to different subcellular regions and organelles. In S. macrospora and N. crassa, the STRIPAK complex is associated with the nuclear envelope, where it might interact with the MAP kinase signaling network [54,60]. The phocein MOB-3 is phosphorylated by MAK-2, and this phosphorylation controls nuclear entry of the MAK-1 kinase, indicating the close integration of these different signaling hubs [54].…”

A novel STRIPAK complex component mediates hyphal fusion and fruiting‐body development in filamentous fungi

“…In previous studies, many factors required for the development of fruiting bodies have been identified in S. macrospora (Kück et al, ; Teichert et al, ). Among these factors, the multiprotein complex striatin‐interacting phosphatase and kinase complex (STRIPAK) (Kück et al, ) has gained significant interest beyond fungal development because its components are conserved from yeasts to mammals (Table ) (Xiang et al, ; Pöggeler and Kück, ; Shim et al, ; Goudreault et al, ; Maerz et al, ; Bloemendal et al, ; Engh et al, ; Simonin et al, ; Wang et al, ; Bernhards and Pöggeler, ; Bloemendal et al, ; Santarpia et al, ; Dettmann et al, ; Nordzieke et al, ; Beier et al, ; Green et al, ; Kück et al, ; Wang et al, ).…”

“…Human STRIPAK complex includes: PP2Ac: Protein phosphatase 2 A catalytic subunit, PP2AA: Protein phosphatase 2 A scaffolding subunit A, MOB3: Monopolar spindle one binder 3, CCM3: Cerebral cavernous malformation 3, STRIP1/2: Striatin interacting proteins 1 and 2, GCK III: Germinal center kinase III family of Ste20 kinases, CTTNBP2/CTTNBP2NL: Cortactin‐binding protein 2, SLMAP: Sarcolemmal membrane‐associated protein, TRAF3IP3: Tumor necrosis factor receptor‐associated factor 3 interacting protein 3, SIKE: Suppressor of IKKε, and FGFR1OP2: Fibroblast growth factor receptor oncogene partner 2. a Identified in this study with BLASTP as SCI1 homolog, b identified in this study. In S. macrospora orthologs of the STRIPAK complex, but not SCI1 were identified and characterized by Beier et al (), Bernhards and Pöggeler (), Bloemendal et al (), Bloemendal et al (), Nordzieke et al (), Pöggeler and Kück () and Kück et al ().…”

“…The components of the STRIPAK complex have also been detected in filamentous ascomycetes. They regulate hyphal fusion, vegetative growth and sexual development (Bloemendal et al, ; Bernhards and Pöggeler, ; Bloemendal et al, ; Nordzieke et al, ; Beier et al, ).…”