Dunking into the Lipid Bilayer: How Direct Membrane Binding of Nucleoporins Can Contribute to Nuclear Pore Complex Structure and Assembly

Nuclear pore complexes (NPCs) mediate the selective and highly efficient transport between the cytoplasm and the nucleus. They are embedded in the two membrane structure of the nuclear envelope at sites where these two membranes are fused to pores. A few transmembrane proteins are an integral part of NPCs and thought to anchor these complexes in the nuclear envelope. In addition, a number of nucleoporins without membrane spanning domains interact with the pore membrane. Here we review our current knowledge of how these proteins interact with the membrane and how this interaction can contribute to NPC assembly, stability and function as well as shaping of the pore membrane.

The First Cytoplasmic Loop in the Core Structure of the ABCC1 (Multidrug Resistance Protein 1; MRP1) Transporter Contains Multiple Amino Acids Essential for Its Expression

ABCC1 (human multidrug resistance protein 1 (hMRP1)) is an ATP-binding cassette transporter which effluxes xeno- and endobiotic organic anions and confers multidrug resistance through active drug efflux. The 17 transmembrane α-helices of hMRP1 are distributed among three membrane spanning domains (MSD0, 1, 2) with MSD1,2 each followed by a nucleotide binding domain to form the 4-domain core structure. Eight conserved residues in the first cytoplasmic loop (CL4) of MSD1 in the descending α-helix (Gly392, Tyr404, Arg405), the perpendicular coupling helix (Asn412, Arg415, Lys416), and the ascending α-helix (Glu422, Phe434) were targeted for mutagenesis. Mutants with both alanine and same charge substitutions of the coupling helix residues were expressed in HEK cells at wild-type hMRP1 levels and their transport activity was only moderately compromised. In contrast, mutants of the flanking amino acids (G392I, Y404A, R405A/K, E422A/D, and F434Y) were very poorly expressed although Y404F, E422D, and F434A were readily expressed and transport competent. Modeling analyses indicated that Glu422 and Arg615 could form an ion pair that might stabilize transporter expression. However, this was not supported by exchange mutations E422R/R615E which failed to improve hMRP1 levels. Additional structures accompanied by rigorous biochemical validations are needed to better understand the bonding interactions crucial for stable hMRP1 expression.

Characterizing the retroviral envelope glycoprotein membrane proximal external region and membrane-spanning domains for their roles in helical alignment, fusogenicity, and incorporation into viral particles.

Viruses use surface proteins to gain access into target cells to initiate infection. How these surface proteins make it into assembling viral particles and the exact details of the entry process are poorly understood. My work here provides insight into how Murine Leukemia Virus (MLV) acquires its surface protein and how this surface protein initiates entry into the target cell. I have identified that two domains in the surface protein dictate its ability to be acquired by the virus. Further, I have identified that four specific amino acid residues in a specific domain of the protein significantly contribute to the entry mechanism

The gene cluster of aureocyclicin 4185: the first cyclic bacteriocin of Staphylococcus aureus

Staphylococcus aureus 4185 was previously shown to produce at least two bacteriocins. One of them is encoded by pRJ101. To detect the bacteriocin-encoding gene cluster, an ~9160 kb region of pRJ101 was sequenced. In silico analyses identified 10 genes (aclX, aclB, aclI, aclT, aclC, aclD, aclA, aclF, aclG and aclH) that might be involved in the production of a novel cyclic bacteriocin named aureocyclicin 4185. The organization of these genes was quite similar to that of the gene cluster responsible for carnocyclin A production and immunity. Four putative proteins encoded by these genes (AclT, AclC, AclD and AclA) also exhibited similarity to proteins encoded by cyclic bacteriocin gene clusters. Mutants derived from insertion of Tn917-lac into aclC, aclF, aclH and aclX were affected in bacteriocin production and growth. AclX is a 205 aa putative protein not encoded by the gene clusters of other cyclic bacteriocins. AclX exhibits 50 % similarity to a permease and has five putative membrane-spanning domains. Transcription analyses suggested that aclX is part of the aureocyclicin 4185 gene cluster, encoding a protein required for bacteriocin production. The aclA gene is the structural gene of aureocyclicin 4185, which shows 65 % similarity to garvicin ML. AclA is proposed to be cleaved off, generating a mature peptide with a predicted M r of 5607 Da (60 aa). By homology modelling, AclA presents four α-helices, like carnocyclin A. AclA could not be found at detectable levels in the culture supernatant of a strain carrying only pRJ101. To our knowledge, this is the first report of a cyclic bacteriocin gene cluster in the genus Staphylococcus.

Mammalian multidrug-resistance proteins (MRPs)

Subfamily C of the human ABC (ATP-binding cassette) superfamily contains nine proteins that are often referred to as the MRPs (multidrug-resistance proteins). The ‘short’ MRP/ABCC transporters (MRP4, MRP5, MRP8 and ABCC12) have a typical ABC structure with four domains comprising two membrane-spanning domains (MSD1 and MSD2) each followed by a nucleotide-binding domain (NBD1 and NBD2). The ‘long’ MRP/ABCCs (MRP1, MRP2, MRP3, ABCC6 and MRP7) have five domains with the extra domain, MSD0, at the N-terminus. The proteins encoded by the ABCC6 and ABCC12 genes are not known to transport drugs and are therefore referred to as ABCC6 and ABCC12 (rather than MRP6 and MRP9) respectively. A large number of molecules are transported across the plasma membrane by the MRPs. Many are organic anions derived from exogenous sources such as conjugated drug metabolites. Others are endogenous metabolites such as the cysteinyl leukotrienes and prostaglandins which have important signalling functions in the cell. Some MRPs share a degree of overlap in substrate specificity (at least in vitro), but differences in transport kinetics are often substantial. In some cases, the in vivo substrates for some MRPs have been discovered aided by studies in gene-knockout mice. However, the molecules that are transported in vivo by others, including MRP5, MRP7, ABCC6 and ABCC12, still remain unknown. Important differences in the tissue distribution of the MRPs and their membrane localization (apical in contrast with basolateral) in polarized cells also exist. Together, these differences are responsible for the unique pharmacological and physiological functions of each of the nine ABCC transporters known as the MRPs.

Assembly and Misassembly of Cystic Fibrosis Transmembrane Conductance Regulator: Folding Defects Caused by Deletion of F508 Occur Before and After the Calnexin-dependent Association of Membrane Spanning Domain (MSD) 1 and MSD2

Cystic fibrosis transmembrane conductance regulator (CFTR) is a polytopic membrane protein that functions as a Cl− channel and consists of two membrane spanning domains (MSDs), two cytosolic nucleotide binding domains (NBDs), and a cytosolic regulatory domain. Cytosolic 70-kDa heat shock protein (Hsp70), and endoplasmic reticulum-localized calnexin are chaperones that facilitate CFTR biogenesis. Hsp70 functions in both the cotranslational folding and posttranslational degradation of CFTR. Yet, the mechanism for calnexin action in folding and quality control of CFTR is not clear. Investigation of this question revealed that calnexin is not essential for CFTR or CFTRΔF508 degradation. We identified a dependence on calnexin for proper assembly of CFTR's membrane spanning domains. Interestingly, efficient folding of NBD2 was also found to be dependent upon calnexin binding to CFTR. Furthermore, we identified folding defects caused by deletion of F508 that occurred before and after the calnexin-dependent association of MSD1 and MSD2. Early folding defects are evident upon translation of the NBD1 and R-domain and are sensed by the RMA-1 ubiquitin ligase complex.