ER network formation requires a balance of the dynamin-like GTPase Sey1p and the Lunapark family member Lnp1p

Abstract

While studies on endoplasmic reticulum (ER) structure and dynamics have focused on the ER tubule forming proteins (reticulons and DP1/Yop1p) and the tubule fusion protein atlastin, nothing is known about the proteins and processes that act to counter-balance this machinery. Here we show that Lnp1p, a member of the conserved lunapark family, plays a role in ER network formation. Lnp1p binds to the reticulons and Yop1p and resides at ER tubule junctions in both yeast and mammalian cells. In the yeast Saccharomyces cerevisiae, the interaction of Lnp1p with the reticulon protein, Rtn1p, and the localization of Lnp1p to ER junctions are regulated by Sey1p, the yeast ortholog of atlastin. We propose that Lnp1p and Sey1p act antagonistically to balance polygonal network formation. In support of this proposal, we show that the collapsed, densely reticulated ER network in lnp1Δ cells is partially restored when the GTPase activity of Sey1p is abrogated.

Arabidopsis Lunapark proteins are involved in ER cisternae formation

“…Yeast and mammalian Lnp proteins preferentially localize to the three‐way junctions of the ER network and it has been suggested that in these organisms Lnp proteins are involved in stabilizing these intersections (Chen et al ., 2012, 2015; Wang et al ., 2016). Although our results showed that AtLNP1 preferentially localizes to ER cisternae rather than junctions, we wanted to determine if AtLNP1 still has a comparable function in stabilizing the overall ER network.…”

“…This is in contrast to the subcellular localization described for yeast and mammalian Lnp proteins which is rather specific to three‐way junctions; yeast and mammalian Lnp proteins label c . 50% of three‐way junctions, resulting in their stabilization (Chen et al ., 2012; Shemesh et al ., 2014; Wang et al ., 2016). Both AtLNP1 and its animal counterpart can also label ER tubules at higher protein expression levels (Shemesh et al ., 2014; Wang et al ., 2016).…”

“…Yeast and mammalian LNP proteins preferentially localize to ER three‐way junctions in vivo and, although they are not required for ER network formation, it has been suggested that LNP acts to stabilize these intersections, potentially acting as temporary scaffold in nascent junctions (Chen et al ., 2012, 2015; Wang et al ., 2016). Immunofluorescence staining of mammalian COS‐7 cells with an anti‐mLnp1 antibody showed that only around half of all three‐way junctions contained mLnp1 but that mLnp1 acquisition was associated with enhanced junction survival probability and reduced junction mobility and ring closure (Chen et al ., 2015).…”

“…Lnp1p has been reported to work synergistically with RTNs and Yop1 (an additional curvature stabilizing protein found in yeast, orthologous to mammalian REEPs), but in antagonism to Sey1p to maintain the cortical ER network in yeast cells (Chen et al ., 2012). Indeed, in sey1∆ yeast mutants lacking functional Sey1p, Lnp1p is expressed in cisternae throughout the ER network, including the nuclear envelope, suggesting that in the wild‐type, Sey1 acts upstream of Lnp1, restricting Lnp1 to ER tubule junctions (Chen et al ., 2012).…”

“…Indeed, in sey1∆ yeast mutants lacking functional Sey1p, Lnp1p is expressed in cisternae throughout the ER network, including the nuclear envelope, suggesting that in the wild‐type, Sey1 acts upstream of Lnp1, restricting Lnp1 to ER tubule junctions (Chen et al ., 2012). Coimmunoprecipitation of epitope‐tagged Lnp1p with RTN1p, Sey1p or Yop1p proteins showed that Lnp1p was capable of interacting with all three yeast ER morphogens in vivo , and that, moreover, the interaction of Lnp1p with Rtn1p is negatively regulated by Sey1p (Chen et al ., 2012). However, more recently Wang et al .…”