Cohen, F. S. | bioRxiv Science

bioRxiv2023

Human proton channels accumulate in cholesterol dependent membrane domains via direct interaction with stomatin

Many membrane proteins are modulated by cholesterol. Here we report strong effects of cholesterol depletion and restoration on the human voltage gated proton channel, hHV1 in excised patches but negligible effects in whole-cell configuration. Despite the presence of a putative cholesterol binding site, a CARC domain in the human voltage gated proton channel, hHV1, mutation of this domain did not affect cholesterol effects. The murine HV1 lacks a CARC sequence but displays similar cholesterol effects. These three results all argue against a direct effect of cholesterol on HV1. We propose that the data are explainable if HV1 preferentially associates with cholesterol-dependent lipid domains, or "rafts." The rafts would be expected to concentrate in the membrane/glass interface and to be depleted from the electrically-accessible patch membrane. This idea is supported by evidence that HV1 channels can diffuse between seal and patch membranes when suction is applied. Suction pulls membrane constituents including HV1 into the patch. In whole-cell studies moderate osmotic stretch does not noticeably alter H+ currents. Simultaneous truncation of the large intracellular N- and C-termini greatly attenuated the cholesterol effect, but C-truncation only did not. We conclude that the N-terminus is the region of attachment to lipid domains. Searching for abundant raft-associated molecules led to stomatin. Co-immunoprecipitation experiments showed that hHV1 binds to stomatin. The stomatin-mediated association of HV1 with cholesterol-dependent lipid domains provides a mechanism for cells to direct HV1 to subcellular location where it is needed, such as the phagosome in leukocytes. SignificanceMany membrane proteins are modulated by cholesterol. Here we explore effects of cholesterol on the human voltage-gated proton channel, hHV1. Although we find little evidence for a direct effect, cholesterol was found to exert a strong influence over H+ current in excised membrane patches. These effects are explainable by hypothesizing that HV1 preferentially associates with cholesterol-dependent membrane lipid domains. We postulate that HV1 diffuses within the membrane and is concentrated in such domains that are anchored to the pipette glass by large membrane proteins. We find that HV1 co-immunoprecipitates with stomatin, a typical component of cholesterol dependent lipid domains. The association of HV1 with lipid domains provides a mechanism for directing HV1 to specific subcellular locations to perform specific functions.

physiology↗

bioRxiv2021

The late endosome-resident lipid bis(monoacylglycero)phosphate is a cofactor for Lassa virus fusion

Arenavirus entry into host cells occurs through a low pH-dependent fusion with late endosomes that is mediated by the viral glycoprotein complex (GPC). The mechanisms of GPC-mediated membrane fusion and of virus targeting to late endosomes are not well understood. To gain insights into arenavirus fusion, we examined cell-cell fusion induced by the Old World Lassa virus (LASV) GPC complex. LASV GPC-mediated cell fusion is more efficient and occurs at higher pH in cells expressing human LAMP1 compared to cells lacking this cognate receptor, but this receptor is not absolutely required for virus entry. GPC-induced fusion progresses through the same lipid intermediates as fusion mediated by other viral glycoproteins - a lipid curvature-sensitive intermediate upstream of hemifusion and a hemifusion intermediate downstream of acid-dependent steps that can be arrested in the cold. Importantly, GPC-mediated fusion is specifically augmented by an anionic lipid, bis(monoacylglycero)phosphate (BMP), which is highly enriched in late endosomes. We show that BMP promotes late steps of LASV fusion downstream of hemifusion - the formation and enlargement of fusion pores. This lipid also specifically promotes cell fusion mediated by GPC of the unrelated New World Junin arenavirus. The BMP-dependence of post-hemifusion stages of arenavirus fusion suggests that these viruses evolved to use this lipid as a cofactor to direct virus entry to late endosomes. Author SummaryPathogenic arenaviruses pose a serious health threat. The viral envelope glycoprotein GPC mediates attachment to host cells and drives virus entry via endocytosis and low pH-dependent fusion within late endosomes. Understanding the host factors and processes that are essential for arenavirus fusion may identify novel therapeutic targets. To delineate the mechanism of arenavirus entry, we examined cell-cell fusion induced by the Old World Lassa virus GPC proteins at low pH. Lassa virus fusion was augmented by the LAMP1 receptor and progressed through lipid curvature-sensitive intermediates, such as hemifusion (merger of contacting leaflets of viral and cell membrane without the formation of a fusion pore). We found that most GPC-mediated fusion events were off-path hemifusion structures and that the transition from hemifusion to full fusion and fusion pore enlargement were specifically promoted by an anionic lipid, bis(monoacylglycero)phosphate, which is highly enriched in late endosomes. This lipid also specifically promotes fusion of unrelated New World Junin arenavirus. Our results imply that arenaviruses evolved to use bis(monoacylglycero)phosphate to enter cells from late endosomes.

microbiology↗