Specificity of Intramembrane Protein–Lipid Interactions

Our concept of biological membranes has markedly changed, from the fluid mosaic model to the current model that lipids and proteins have the ability to separate into microdomains, differing in their protein and lipid compositions. Since the breakthrough in crystallizing membrane proteins, the most powerful method to define lipid-binding sites on proteins has been X-ray and electron crystallography. More recently, chemical biology approaches have been developed to analyze protein–lipid interactions. Such methods have the advantage of providing highly specific cellular probes. With the advent of novel tools to study functions of individual lipid species in membranes together with structural analysis and simulations at the atomistic resolution, a growing number of specific protein–lipid complexes are defined and their functions explored. In the present article, we discuss the various modes of intramembrane protein–lipid interactions in cellular membranes, including examples for both annular and nonannular bound lipids. Furthermore, we will discuss possible functional roles of such specific protein–lipid interactions as well as roles of lipids as chaperones in protein folding and transport.Our concept of biological membranes has markedly changed in the last two decades, from the fluid mosaic model (Singer and Nicolson 1972), in which the membrane was thought to be formed by a homogenous lipid fluid phase with proteins embedded, to the current model that lipids and proteins are not homogenously distributed, but have the ability to separate into microdomains, differing in their protein and lipid compositions. A well established example of domains are lipid rafts (see Box 1 for definitions). Raft domains are described as dynamic domain structures enriched in cholesterol, sphingolipids, and membrane proteins (Brown and London 1998; Simons and Ikonen 1997) that have an important role in different cellular processes (Lingwood and Simons 2010). Formation of domains within cellular membranes has been extensively investigated over the past years leading to various models that differ in the primary forces involved in the formation and the recruitment of surrounding membrane components into such domains.

BOX 1.

Definitions

Annular Lipids/Lipid Shell

An annular lipid shell is formed when selected lipid classes or molecular species bind preferentially to the hydrophobic and/or hydrophilic surfaces of a membrane protein. Per definition these lipids show markedly reduced residence times at the protein–lipid interface as compared to bulk lipids.

Bulk Lipids

Lipids within the membrane that diffuse rapidly in the bilayer plane and show a low residence time at the protein–lipid interface following random collisions. Typical diffusion coefficients for bulk lipids in a liquid disordered phase are in the range of D_L = 7×10⁻¹² m²/sec (DOPC) (Filippov et al. 2003).

Hydrophobic Mismatch

A term to describe any deviation from the compatibility of the hydrophobic surface of membrane proteins (their TMDs) to the vertically and laterally encountered hydrophobic surfaces of the lipid bilayer in biological membranes. In the case of a hydrophobic mismatch, the resulting energy penalty may cause the recruitment of a suitable local lipid environment, the deformation of the membrane and/or in conformational changes of the protein to achieve a status of hydrophobic match (for advanced reading, see Killian 1998).

Lateral Pressure Field/Profile of Membranes

Biological membranes can be considered as the “solvent” for membrane proteins that are embedded in them. The lateral pressure profile (Ω(z)) describes the force or pressure that is exerted by the membrane on the matter residing inside it. This pressure is modulated by different extents of lipid–lipid interactions and asymmetries across and within the bilayer, which in turn results in varying lateral pressures that may locally correspond to several hundreds of atmospheres.

Lipid Rafts

Sterol and sphingolipid-dependent microdomains that form a network of lipid–lipid, protein–protein, and protein–lipid interactions; involved in the compartmentalization of processes such as signaling within biological membranes.

Liquid-Disordered Phase (I_d)

A predominantly fluid phase of lipids, characterized by a high degree of mobility (cis-gauche flexibility of acyl chains; lateral diffusion) and a high content of short and/or unsaturated fatty acyl chains.

Liquid-Ordered Phase (I_o)

A liquid crystalline phase (that displays physical properties of both liquids and of solid crystals), characterized by a high degree of acyl chain order (“packing”), a reduced lateral mobility of lipid and protein molecules, and a reduction in the elasticity of the membrane as a result of specific interactions between sterols and phospholipids containing long, saturated acyl chains and/or glycosphingolipids.

Microdomains

Membrane compartments of distinct lipid and protein composition that may modulate the enzymatic functions of membrane proteins.

Molecular Lipid Species

Individual members of a lipid class that differ in their fatty acid composition.

Nonannular Lipids

Lipids that specifically interact with membrane proteins are neither bulk lipids, nor do they belong to the shell/annulus of lipids that surround the membrane protein. These nonannular lipids often reside within membrane protein complexes, in which they may fulfill diverse functions ranging from structural building blocks to allosteric effectors of enzymatic activity (see text). Nonannular lipids bind to distinct hydrophobic sites of membrane proteins or membrane protein complexes.According to one model, membrane domains can form by specific protein–protein interactions (Douglass and Vale 2005). This model is based on single-molecule microscopy experiments. In these studies, single fluorophores were chemically attached to specific proteins, and the dynamics of individual proteins was tracked by monitoring the fluorescent probe. In this kind of set up, a dynamic behavior of lipids is not assessed. Here, proteins involved in signaling processes are trapped within interconnected microdomains created by specific protein–protein interactions, probably involving additional scaffolding proteins. The proteins of such domains can exchange with the surrounding membrane area at individual kinetics, some components are immobile over minutes, and others can diffuse rapidly.Another model emphasizes the importance of lipid–lipid interactions, initiating the formation of subdomains of defined lipid compositions. Transmembrane proteins then can be attracted to such subdomains via various specific interactions with lipids. The resulting lipid–protein complexes then eventually coalesce to form larger lipid–protein assemblies (Anderson and Jacobson 2002).The idea of lipid-dependent domain formation is inherent to the biophysical properties and therefore to the complex lipid composition of cellular membranes that include up to a thousand lipids that vary in structure (van Meer et al. 2008). This wide range of lipid species has been proposed to facilitate the “solvation” of membrane proteins. Taken into account the sum of lipid species present in a cellular membrane, it is important to understand the different interactions and affinities within the bilayer between different lipids. Molecular dynamics simulations have been successfully employed to investigate lipid interactions between different lipid species and found specific interactions of various lipid classes and molecular species (Hofsass et al. 2003; Niemela et al. 2004, 2006, 2009; Pandit et al. 2004; Zaraiskaya and Jeffrey 2005; Bhide et al. 2007). These results are supported and expanded by recent data from our group that suggest a specific order of interactions of sphingomyelin species with cholesterol in membranes (A.M. Ernst, F. Wieland, and B. Brügger, unpubl.). At low cholesterol concentrations, some sphingomyelin species preferentially interact with cholesterol, whereas others prefer their kin. At higher cholesterol concentrations, all sphingomyelin species investigated display an increased affinity for the sterol. These findings open the possibility of differentiated pathways of self-assembly of microdomains, dependent on molecular lipid species.In the present article the various modes of intramembrane protein–lipid interactions in cellular membranes (Fig. 1) will be discussed. This includes possible functional roles of such specific protein–lipid interactions.Open in a separate windowFigure 1.Intramembrane protein–lipid interactions within a cell membrane. (A) Bulk lipids; (B) annular lipids; (C) nonannular lipids/lipid ligands. For details see text.     

The Interfacial Tension of the Lipid Membrane Formed from Lipid–Amino Acid Systems

The interfacial tension of lipid membranes composed of phosphatidylcholine (lecithin, PC)–valine (Val), phosphatidylcholine–isoleucine (Ile), phosphatidylcholine–tyrosine (Tyr), and phosphatidylcholine–phenylalanine (Phe) has been studied. The membrane components formed 1:1 complexes. The interfacial tension measurements were used to determine the membrane surface concentration A ₃⁻¹, the membrane interfacial tension γ₃, and the stability constant K.     

Lipid modifications of proteins – slipping in and out of membranes

Protein lipid modification, once thought to act as a stable membrane anchor for soluble proteins, is now attracting more widespread attention for its emerging role in diverse signaling pathways and regulatory mechanisms. Most multicellular organisms have recruited specific types of lipids and a suite of unique enzymes to catalyze the modification of a select number of proteins, many of which are evolutionarily conserved in plants, animals and fungi. Each of the three known types of lipid modification – palmitoylation, myristylation and prenylation – allows cells to target proteins to the plasma membrane, as well as to other subcellular compartments. Among the lipid modifications, protein prenylation might also function as a relay between cytoplasmic isoprene biosynthesis and regulatory pathways that control cell cycle and growth. Molecular and genetic studies of an Arabidopsis mutant that lacks farnesyl transferase suggest that the enzyme has a role in abscisic acid signaling during seed germination and in the stomata. It is becoming clear that lipid modifications are not just fat for the protein, but part of a highly conserved intricate network that plays a role in coordinating complex cellular functions.     

Clustering of α-Synuclein on Supported Lipid Bilayers: Role of Anionic Lipid, Protein, and Divalent Ion Concentration

α-Synuclein is the major component of Lewy body inclusions found in the brains of patients with Parkinson's disease. Several studies indicate that α-synuclein binds to negatively charged phospholipid bilayers. We examined the binding of α-synuclein to membranes containing different amounts of negatively charged lipids using supported lipid bilayers, epifluorescence microscopy, fluorescence recovery after photobleaching, and bulk fluorescence techniques. The membranes contained phosphatidylcholine and phosphatidylglycerol. In the absence of protein, these lipids mix uniformly. Our results show that the propensity of α-synuclein to cluster on the membrane increases as the concentration of anionic lipid and/or protein increases. Regions on the lipid bilayer where α-synuclein is clustered are enriched in phosphatidylglycerol. We also observe divalent metal ions stimulate protein cluster formation, primarily by promoting lipid demixing. The importance of protein structure, lipid demixing, and divalent ions, as well as the physiological implications, will be discussed. Because membrane-bound α-synuclein assemblies may play a role in neurotoxicity, it is of interest to determine how membranes can be used to tune the propensity of α-synuclein to aggregate.     

Lipid analysis of the plasma membrane and mitochondria of brewer’s yeast

The plasma membrane and mitochondria of bottom fermenting brewer's yeast obtained as a by-product of industrial beer production were isolated and the lipid fraction was analyzed. The phospholipid content accounted for 78 mg/g protein in the plasma membrane and 59 mg/g protein in the mitochondria. Major phospholipids in both preparations were phosphatidylinositol, phosphatidylcholine and phosphatidylethanolamine but their proportions differed significantly. In the plasma membrane phosphatidylinositol, and in the mitochondria phosphatidylcholine were present in the highest concentration (37 and 30%, respectively). The main classes of neutral lipids (triacylglycerols, ergosterol, squalene and steryl esters) were twice more abundant in the plasma membrane than in the mitochondria (61 and 33 mg/g protein, respectively). A characteristic of the neutral lipid composition of both organelles was the low content of ergosterol (12 and 7 mg/g protein, respectively) and a high content of squalene (25 and 22 mg/g protein). The main feature of the fatty acid composition of both organelles was the preponderance of saturated fatty acids (78 and 79%, respectively), among which palmitic acid was the principal one. The most expressed characteristics of lipid fractions of the analyzed plasma membranes and mitochondria, high concentration of squalene and preponderance of saturated fatty acids are the consequences of anaerobic growth conditions. The lack of oxygen had possibly the strongest effect on the lipid composition of the plasma membranes and mitochondria of bottom fermenting brewer's yeast.     

Lipid and phase specificity of α-toxin from S. aureus

The pore forming toxin Hla (α-toxin) from Staphylococcus aureus is an important pathogenic factor of the bacterium S. aureus and also a model system for the process of membrane-induced protein oligomerisation and pore formation. It has been shown that binding to lipid membranes at neutral or basic pH requires the presence of a phosphocholine-headgroup. Thus, sphingomyelin and phosphatidylcholine may serve as interaction partners in cellular membranes. Based on earlier studies it has been suggested that rafts of sphingomyelin are particularly efficient in toxin binding. In this study we compared the oligomerisation of Hla on liposomes of various lipid compositions in order to identify the preferred interaction partners and conditions. Hla seems to have an intrinsic preference for sphingomyelin compared to phosphatidylcholine due to a higher probability of oligomerisation of membrane bound monomer. We also can show that increasing the surface density of Hla-binding sites enhances the oligomerisation efficiency. Thus, preferential binding to lipid rafts can be expected in the cellular context. On the other hand, sphingomyelin in the liquid disordered phase is a more favourable binding partner for Hla than sphingomyelin in the liquid ordered phase, which makes the membrane outside of lipid rafts the more preferred region of interaction. Thus, the partitioning of Hla is expected to strongly depend on the exact composition of raft and non-raft domains in the membrane.     

Lipid binding specificity of bovine α-lactalbumin: A multidimensional approach

Many soluble proteins are known to interact with membranes in partially disordered states, and the mechanism and relevance of such interactions in cellular processes are beginning to be understood. Bovine α-lactalbumin (BLA) represents an excellent prototype for monitoring membrane interaction due to its conformational plasticity. In this work, we comprehensively monitored the interaction of apo-BLA with zwitterionic and negatively charged membranes utilizing a variety of approaches. We show that BLA preferentially binds to negatively charged membranes at acidic pH with higher binding affinity. This is supported by spectral changes observed with a potential-sensitive membrane probe and fluorescence anisotropy measurements of a hydrophobic probe. Our results show that BLA exhibits a molten globule conformation when bound to negatively charged membranes. We further show, using the parallax approach, that BLA penetrates the interior of negatively charged membranes, and tryptophan residues are localized at the membrane interface. Red edge excitation shift (REES) measurements reveal that the immediate environment of tryptophans in membrane-bound BLA is restricted, and the restriction is dependent on membrane lipid composition. We envision that understanding the mechanism of BLA–membrane interaction would help in bioengineering of α-lactalbumin, and to address the mechanism of tumoricidal and antimicrobial activities of BLA–oleic acid complex.     

Lipid peroxidation as the mechanism of modification of brain 5′-nucleotidase activity in vitro

The effect of lipid peroxidation on the Mg²⁺-independent and Mg²⁺-dependent activity of brain cell membrane 5-nucleotidase was determined and the affinity of the active sites of Mg²⁺-dependent enzyme for 5-AMP (substrate) and Mg²⁺ (activator) was examined. Brain cell membranes were peroxidized at 37°C in the presence of 100 M ascorbate and 25 M FeCl₂ (resultant) for 10 min. The activity of 5-nucleotidase and lipid peroxidation products (thiobarbituric acid reactive substances) were determined. At 10 min, the level of lipid peroxidation products increased from 0.20±0.10 to 17.5±1.5 nmoles malonaldehyde/mg membrane protein. The activity of Mg²⁺-independent 5-nucleotidase increased from 0.201±0.020 in controls to 0.305±0.028 mol Pi/mg protein/hr in peroxidized membranes. In the presence of 10mM Mg²⁺, the activity increased by 5.8-fold in the peroxidized membrane preparation in comparison to 14-fold in control In peroxidized preparation, the affinity of active site of Mg²⁺-dependent 5-nucleotidase for 5-AMP tripled, as indicated by a significant decrease inK _m (K _m=95±2 M AMP for control;K _m=32±2 MAMP for peroxidized).V _max was significantly reduced from 3.35±0.16 in control to 1.70±.09 moles Pi/mg protein in peroxidized membranes. The affinity of the active site for Mg²⁺ significantly increased (K _m=6.17±0.37 mM Mg²⁺ for control;K _m=4.0±0.31 peroxidized). The data demonstrate that lipid peroxidation modifies the Mg²⁺-dependent 5-nucleotidase function by altering the active sites for both the substrate and the activator. The modification of the 5-nucleotidase activity and the loss of Mg²⁺-dependent activation observed in this in-vitro study are similar to the changes previously observed by us in the hypoxic brain in-vivo. This suggests that lipid peroxidation which specifically alters the active site may be the underlying mechanism of the modification of 5-nucleotidase during hypoxia.     

Lipid composition influences the release of Alzheimer's amyloid β-peptide from membranes

The behavior of the amyloid β-peptide (Aβ) within a membrane environment is integral to its toxicity and the progression of Alzheimer's disease. Ganglioside GM1 has been shown to enhance the aggregation of Aβ, but the underlying mechanism is unknown. Using atomistic molecular dynamics simulations, we explored the interactions between the 40-residue alloform of Aβ (Aβ(40) ) and several model membranes, including pure palmitoyloleoylphosphatidylcholine (POPC) and palmitoyloleoylphosphatidylserine (POPS), an equimolar mixture of POPC and palmitoyloleoylphosphatidylethanolamine (POPE), and lipid rafts, both with and without GM1, to understand the behavior of Aβ(40) in various membrane microenvironments. Aβ(40) remained inserted in POPC, POPS, POPC/POPE, and raft membranes, but in several instances exited the raft containing GM1. Aβ(40) interacted with GM1 largely through hydrogen bonding, producing configurations containing β-strands with C-termini that, in some cases, exited the membrane and became exposed to solvent. These observations provide insight into the release of Aβ from the membrane, a previously uncharacterized process of the Aβ aggregation pathway.     

Lipid Integration in Neurodegeneration: An Overview of Alzheimer’s Disease

Various types of lipids and their metabolic products associated with the biological membrane play a crucial role in signal transduction, modulation, and activation of receptors and as precursors of bioactive lipid mediators. Dysfunction in the lipid homeostasis in the brain could be a risk factor for the many types of neurodegenerative disorders, including Alzheimer’s disease, Huntington’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis. These neurodegenerative disorders are marked by extensive neuronal apoptosis, gliosis, and alteration in the differentiation, proliferation, and development of neurons. Sphingomyelin, a constituent of plasma membrane, as well as its primary metabolite ceramide acts as a potential lipid second messenger molecule linked with the modulation of various cellular signaling pathways. Excessive production of reactive oxygen species associated with enhanced oxidative stress has been implicated with these molecules and involved in the regulation of a variety of different neurodegenerative and neuroinflammatory disorders. Studies have shown that alterations in the levels of plasma lipid/cholesterol concentration may result to neurodegenerative diseases. Alteration in the levels of inflammatory cytokines and mediators in the brain has also been found to be implicated in the pathophysiology of neurodegenerative diseases. Although several mechanisms involved in neuronal apoptosis have been described, the molecular mechanisms underlying the correlation between lipid metabolism and the neurological deficits are not clearly understood. In the present review, an attempt has been made to provide detailed information about the association of lipids in neurodegeneration especially in Alzheimer’s disease.     

Conserved Amphipathic Helices Mediate Lipid Droplet Targeting of Perilipins 1–3

Perilipins (PLINs) play a key role in energy storage by orchestrating the activity of lipases on the surface of lipid droplets. Failure of this activity results in severe metabolic disease in humans. Unlike all other lipid droplet-associated proteins, PLINs localize almost exclusively to the phospholipid monolayer surrounding the droplet. To understand how they sense and associate with the unique topology of the droplet surface, we studied the localization of human PLINs in Saccharomyces cerevisiae, demonstrating that the targeting mechanism is highly conserved and that 11-mer repeat regions are sufficient for droplet targeting. Mutations designed to disrupt folding of this region into amphipathic helices (AHs) significantly decreased lipid droplet targeting in vivo and in vitro. Finally, we demonstrated a substantial increase in the helicity of this region in the presence of detergent micelles, which was prevented by an AH-disrupting missense mutation. We conclude that highly conserved 11-mer repeat regions of PLINs target lipid droplets by folding into AHs on the droplet surface, thus enabling PLINs to regulate the interface between the hydrophobic lipid core and its surrounding hydrophilic environment.     

Protein Kinase C: The “Masters” of Calcium and Lipid

The coordinated and physiological behavior of living cells in an organism critically depends on their ability to interact with surrounding cells and with the extracellular space. For this, cells have to interpret incoming stimuli, correctly process the signals, and produce meaningful responses. A major part of such signaling mechanisms is the translation of incoming stimuli into intracellularly understandable signals, usually represented by second messengers or second-messenger systems. Two key second messengers, namely the calcium ion and signaling lipids, albeit extremely different in nature, play an important and often synergistic role in such signaling cascades. In this report, we will shed some light on an entire family of protein kinases, the protein kinases C, that are perfectly designed to exactly decode these two second messengers in all of their properties and convey the signaling content to downstream processes within the cell.Once generated, second messengers relay their information content in a plethora of properties, including time, quantity (i.e., concentration), space (i.e., subcellular distribution), and interestingly into any combination of these three characteristics. Nevertheless, such information is meaningless for the cell unless it has a toolkit of read-out systems that can actually interpret such second-messenger properties and relate them further downstream into complex signaling networks, or directly to effector systems. An important system is the family of protein kinase Cs (PKCs) that can read-out lipid signals alone, or combine the ability to read-out simultaneous lipid and Ca²⁺ signals. A common denominator of all PKCs is the property to convey signals downstream by phosphorylation of additional signaling partners or effector proteins. We will briefly introduce the PKC subfamilies with particular emphasis on their signaling ability, discuss the important sensing domains, and their properties, before concentrating on sensing details of the subfamily of conventional PKCs and their role in signal integration in greater depth.     

Lipid peroxidation in chronic gastritis; any influence of Helicobacter pylori?

In an attempt to investigate the significance of lipid peroxidation in the pathogenesis of gastritis associated with or without Helicobacter pylori infection, malonodialdehyde (MDA) levels were measured by the thiobarbiturate assay in the gastric juice of 101 patients undergoing upper GI endoscopy and correlated with histopathological findings. Elevated MDA levels were found in all patients with gastritis compared with controls. MDA levels were significantly correlated with the extent of the mucosal inflammation and with disease activity in patients with reactive gastritis. In patients with H. pylori associated gastritis MDA levels were not correlated with disease activity but rather with the degree of atrophy. In this case, MDA levels were equal or even less than in patients with reactive gastritis. MDA levels were not affected by the history of consumption of PPIs, of H(2)-blockers or of NSAIDs over the last month before the endoscopy. It is concluded that lipid peroxidation is a mechanism involved in the pathogenesis of gastritis associated or not to H. pylori infection.     

Lipid peroxidation in microsomes of murine bone marrow after low-doseγ-irradiation

The principal aim of the study was to investigate the effect of low-dose-irradiation on lipid peroxidation (LPO) in murine bone marrow. To this end, the degree of LPO in suspensions of microsomes of murine bone marrow cells (BMC) was determined in terms of malondialdehyde (MDA) formation after whole-body or in vitro exposure to various doses of-radiation. These effects were compared to some extent with similar effects in liver and spleen preparations. As to the effect of-irradiation on LPO in BMC, the response depends on the dose level and on whether whole-body or in vitro exposures are involved. Whole-body irradiation did not result in an increase in LPO in BMC microsomes, even at such high doses as 15 Gy, although hepatic microsomes showed a marked increase. In contrast, in vitro irradiation of BMC microsomes with 0.1, 10 and 50 Gy brought about an increase in LPO. This increase was already significant (P <0.05) at 0.1 Gy following a post-irradiation incubation and substantial at 50 Gy, even without subsequent incubation. The results show that low doses of-irradiation are able to induce an elevation of LPO in murine BMC microsomes, but only after in vitro irradiation. In the case of whole-body irradiation cellular radical scavengers and other metabolic reactions may prevent a measurable increase in LPO. This is partly illustrated by the case of vitamin-E deficiency, where a substantial increase in LPO in BMC microsomes is observed even without-irradiation in comparison with euvitaminotic mice because normally occurring radicals are not scavenged sufficiently.On leave from the University of Rochester, School of Medicine and Dentistry, Rochester, NY 14678, USA     

Radiating Amyloid Fibril Formation on the Surface of Lipid Membranes through Unit-Assembly of Oligomeric Species of α-Synuclein

Background

Lewy body in the substantia nigra is a cardinal pathological feature of Parkinson''s disease. Despite enormous efforts, the cause-and-effect relationship between Lewy body formation and the disorder is yet to be explicitly unveiled.

Methodology/Principal Findings

Here, we showed that radiating amyloid fibrils (RAFs) were instantly developed on the surface of synthetic lipid membranes from the β-sheet free oligomeric species of α-synuclein through a unit-assembly process. The burgeoning RAFs were successfully matured by feeding them with additional oligomers, which led to concomitant dramatic shrinkage and disintegration of the membranes by pulling off lipid molecules to the extending fibrils. Mitochondria and lysosomes were demonstrated to be disrupted by the oligomeric α-synuclein via membrane-dependent fibril formation.

Conclusion

The physical structure formation of amyloid fibrils, therefore, could be considered as detrimental to the cells by affecting membrane integrity of the intracellular organelles, which might be a molecular cause for the neuronal degeneration observed in Parkinson''s disease.     

Lipid metabolism. Evidence of a δ-oxidation pathway for saturated fatty acids

1. Specific radioactivities of milk triglyceride fatty acids and gamma- and delta-hydroxy fatty acids were measured after the intramammary infusion of [1-(14)C]acetate, delta-hydroxy[1-(14)C]laurate and [1-(14)C]laurate as their sodium salts into fed lactating goats. 2. Net incorporations of the radioactive tracer into the total milk lipids were comparable, being 16, 17 and 21% of the label infused respectively. 3. The specific radioactivities of the C(4)-C(8) fatty acids after [1-(14)C]acetate infusion were lower than those of the C(10)-C(14) fatty acids. 4. After delta-hydroxy[1-(14)C]laurate administration the milk triglyceride fatty acids were labelled and their specific radioactivities were characterized by decreasing values with increasing chain length of the fatty acids, implicating C(4) unit incorporation. 5. The gamma- and delta-hydroxy fatty acids isolated after [1-(14)C]laurate infusion were highly labelled and the milk triglyceride fatty acids, other than laurate, exhibited a labelling pattern similar to that of the fatty acids derived from the radioactive delta-hydroxy fatty acid. 6. Evidence is presented for the existence of saturated fatty acid delta-oxidation in the mammary gland, in which the gamma- and delta-hydroxy fatty acids are active intermediates.