Abstract
VAPB is a ubiquitously expressed, ER-resident adaptor protein involved in interorganellar lipid exchange, membrane contact site formation, and membrane trafficking. Its mutant form, P56S-VAPB, which has been linked to a dominantly inherited form of Amyotrophic Lateral Sclerosis (ALS8), generates intracellular inclusions consisting in restructured ER domains whose role in ALS pathogenesis has not been elucidated. P56S-VAPB is less stable than the wild-type protein and, at variance with most pathological aggregates, its inclusions are cleared by the proteasome. Based on studies with cultured cells overexpressing the mutant protein, it has been suggested that VAPB inclusions may exert a pathogenic effect either by sequestering the wild-type protein and other interactors (loss-of-function by a dominant negative effect) or by a more general proteotoxic action (gain-of-function). To investigate P56S-VAPB degradation and the effect of the inclusions on proteostasis and on ER-to-plasma membrane protein transport in a more physiological setting, we used stable HeLa and NSC34 Tet-Off cell lines inducibly expressing moderate levels of P56S-VAPB. Under basal conditions, P56S-VAPB degradation was mediated exclusively by the proteasome in both cell lines, however, it could be targeted also by starvation-stimulated autophagy. To assess possible proteasome impairment, the HeLa cell line was transiently transfected with the ERAD (ER Associated Degradation) substrate CD3δ, while autophagic flow was investigated in cells either starved or treated with an autophagy-stimulating drug. Secretory pathway functionality was evaluated by analyzing the transport of transfected Vesicular Stomatitis Virus Glycoprotein (VSVG). P56S-VAPB expression had no effect either on the degradation of CD3δ or on the levels of autophagic markers, or on the rate of transport of VSVG to the cell surface. We conclude that P56S-VAPB inclusions expressed at moderate levels do not interfere with protein degradation pathways or protein transport, suggesting that the dominant inheritance of the mutant gene may be due mainly to haploinsufficiency.
Highlights
VAPB, and its homologue VAPA, are members of the highly conserved and ubiquitously expressed VAP (Vesicle-Associated Membrane Protein (VAMP)-Associated Protein) family of ER tailanchored transmembrane proteins
The cytosolic N-terminal region, consists of a domain that is homologous to the nematode major sperm protein (MSP), followed by a central coiled-coil domain; the transmembrane segment is close to the C-terminus, and the last four C-terminal residues are probably exposed to the ER lumen [1]
To investigate the mechanism of P56S-VAPB clearance, we used the previously characterized HeLa Tet-Off cell line [24,25], in which expression of mutant, myc-tagged, VAPB is repressed by tetracycline or Dox, and induced by removal of the antibiotic from the medium
Summary
VAPB, and its homologue VAPA, are members of the highly conserved and ubiquitously expressed VAP (Vesicle-Associated Membrane Protein (VAMP)-Associated Protein) family of ER tailanchored transmembrane proteins. The identification of a dominant missense mutation in the VAPB gene in patients affected by a slowly progressing form of familial motor neuron disease (ALS8) [15] greatly increased the interest in VAP proteins. Identified in eight Brazilian families with a shared Portuguese ancestor [19], the same mutation was subsequently detected in an unrelated German patient, carrying a haplotype distinct from the one linked to the mutation in the Brazilian families [20]. Three additional mutations of VAPB have since been identified in familial Amyotrophic Lateral Sclerosis (ALS) patients [21,22,23], in these cases, the segregation of the mutation with the disease was not demonstrated
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