Active Nuclear Import of Membrane Proteins Revisited.

It is poorly understood how membrane proteins destined for the inner nuclear membrane pass the crowded environment of the Nuclear Pore Complex (NPC). For the Saccharomyces cerevisiae proteins Src1/Heh1 and Heh2, a transport mechanism was proposed where the transmembrane domains diffuse through the membrane while the extralumenal domains encoding a nuclear localization signal (NLS) and intrinsically disordered linker (L) are accompanied by transport factors and travel through the NPC. Here, we validate the proposed mechanism and explore and discuss alternative interpretations of the data. First, to disprove an interpretation where the membrane proteins become membrane embedded only after nuclear import, we present biochemical and localization data to support that the previously used, as well as newly designed reporter proteins are membrane-embedded irrespective of the presence of the sorting signals, the specific transmembrane domain (multipass or tail anchored), independent of GET, and also under conditions that the proteins are trapped in the NPC. Second, using the recently established size limit for passive diffusion of membrane proteins in yeast, and using an improved assay, we confirm active import of polytopic membrane protein with extralumenal soluble domains larger than those that can pass by diffusion on similar timescales. This reinforces that NLS-L dependent active transport is distinct from passive diffusion. Thirdly, we revisit the proposed route through the center of the NPC and conclude that the previously used trapping assay is, unfortunately, poorly suited to address the route through the NPC, and the route thus remains unresolved. Apart from the uncertainty about the route through the NPC, the data confirm active, transport factor dependent, nuclear transport of membrane-embedded mono- and polytopic membrane proteins in baker's yeast.

Traffic to the inner membrane of the nuclear envelope.

Past research has yielded valuable insight into the mechanisms that regulate the nuclear transport of soluble molecules like transcription factors and mRNA. Much less is known about the mechanisms responsible for the transportation of membrane proteins to the inner membrane of the nuclear envelope. The key question is: does the facilitated transport of integral inner membrane proteins exist in the same way as it does for soluble proteins and, if so, what is it used for? Herein, we provide an overview of the current knowledge on traffic to the inner nuclear membrane, and make a case that: (a) known sorting signals and molecular mechanisms in membrane protein biogenesis, membrane protein traffic and nuclear transport are also relevant with respect to INM traffic; and (b) the interplay of the effects of these signals and molecular mechanisms is what determines the rates of traffic to the INM.

The regulatory inputs controlling pleiotropic drug resistance and hypoxic response in yeast converge at the promoter of the aminocholesterol resistance gene RTA1.

Aminosterols possessing potent fungicidal activity are attractive alternatives to currently available antifungals. Although their precise mechanism of action is not fully understood, the effect of 7-aminocholesterol (7-ACH) involves a partial block of Δ8-Δ7 isomerase and C-14 reductase. The function of RTA1 encoding the 7-transmembrane helix protein, cloned as the multicopy suppressor of 7-ACH toxicity in yeast, remains unclear. In this report, we show that Rta1p is localized in the plasma membrane and has a high rate of metabolic turnover, as revealed by fluorescence microscopy, cell fractionation and pulse-chase experiments. Analysis of the RTA1-lacZ reporter activity and deletion mapping of the promoter allowed the identification of the regions responsible for negative regulation by Tup1 and the two synergistically acting repressors of hypoxic genes, Rox1p and Mot3p. This was in line with increased RTA1-mediated resistance to 7-ACH under hypoxic conditions, associated with increased Rta1p level. Overexpression of RTA1 also affected the response to the signalling sphingolipid precursor phytosphingosine. Positive inputs of two transcriptional activators Pdr1p and Upc2p were also detected, indicating a regulatory link common to sterol biosynthetic genes as well as those involved in pleiotropic drug resistance and sphingolipid metabolism.

Long unfolded linkers facilitate membrane protein import through the nuclear pore complex.

Active nuclear import of soluble cargo involves transport factors that shuttle cargo through the nuclear pore complex (NPC) by binding to phenylalanine-glycine (FG) domains. How nuclear membrane proteins cross through the NPC to reach the inner membrane is presently unclear. We found that at least a 120-residue-long intrinsically disordered linker was required for the import of membrane proteins carrying a nuclear localization signal for the transport factor karyopherin-α. We propose an import mechanism for membrane proteins in which an unfolded linker slices through the NPC scaffold to enable binding between the transport factor and the FG domains in the center of the NPC.